Glycosylated specificity exchangers

ABSTRACT

The present invention is directed to ligand/receptor and antigen/antibody specificity exchangers comprising a saccharide or glycoconjugate. Methods of making these specificity exchangers and methods of using said specificity exchangers to treat or prevent human disease are described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityof U.S. patent application Ser. No. 10/773,628, filed Feb. 5, 2004,which claims the benefit of priority of U.S. Provisional PatentApplication No. 60/446,172, filed Feb. 6, 2003. Priority to both of U.S.patent application Ser. No. 10/773,628 and U.S. Provisional PatentApplication No. 60/446,172 is hereby claimed and both applications arehereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to compositions and methods forpreventing and treating human diseases, including cancer, and thoseresulting from pathogens such as bacteria, yeast, parasites, fungus,viruses, and the like. More specifically, embodiments described hereinconcern the manufacture and use of specificity exchangers comprisingglycosylated antigenic domains, which redirect natural antibodies thatare present in a subject to a pathogen.

BACKGROUND OF THE INVENTION

Specificity exchangers are generally composed of two domains, aspecificity domain and an antigenic domain. There are two general typesof specificity exchangers differentiated by the nature of theirspecificity domains. (See e.g., U.S. patent application Ser. No.10/372,735, hereby expressly incorporated by reference in its entirety).The first type of specificity exchanger is an antigen/antibodyspecificity exchanger. Several different types of antigen/antibodyspecificity exchangers can be made. (See e.g., U.S. Pat. Nos. 5,869,232;6,040,137; 6,245,895; 6,417,324; 6,469,143; and U.S. application Ser.Nos. 09/839,447 and 09/839,666; and International App. Nos.PCT/SE95/00468 and PCT/IB01/00844, all of which are hereby expresslyincorporated by reference in their entireties).

Antigen/antibody specificity exchangers comprise an amino acid sequenceof an antibody that specifically binds to an antigen (i.e., thespecificity domain) joined to an amino acid sequence to which anantibody binds (i.e. the antigenic domain). Some specificity domains ofantigen/antibody specificity exchangers comprise an amino acid sequenceof a complementarity determining region (CDR), are at least 5 and lessthan 35 amino acids in length, are specific for HIV-1 antigens, or arespecific for hepatitis viral antigens. Some antigenic domains ofantigen/antibody specificity exchangers comprise a peptide having anantibody-binding region of viral, bacterial, or fungal origin, are atleast 5 and less than 35 amino acids in length, or contain peptides(e.g., peptides comprising epitopes) that are obtained from polio virus,measles virus, hepatitis B virus, hepatitis C virus, or HIV-1.

A second type of specificity exchanger, the ligand/receptor specificityexchanger, is also composed of a specificity domain and an antigenicdomain, however, the specificity domain of the ligand/receptorspecificity exchanger comprises a ligand for a receptor that is presenton a pathogen, as opposed to a sequence of an antibody that binds to anantigen. That is, a ligand/receptor specificity exchanger differs froman antibody/antigen specificity exchanger in that the ligand/receptorspecificity exchanger does not contain a sequence of an antibody thatbinds an antigen but, instead, adheres to the pathogen vis a vis ligandinteraction with a receptor that is present on the pathogen. Severaldifferent types of ligand/receptor specificity exchangers can be made.(See e.g., U.S. Pat. No. 6,660,842; U.S. application Ser. No.10/372,735; and International App. No. PCT/IB01/02327, all of which arehereby expressly incorporated by reference in their entireties).

Some specificity domains of ligand/receptor specificity exchangerscomprise an amino acid sequence that is a ligand for a bacterialadhesion receptor (e.g., extracellular fibrinogen binding protein orclumping factor A or B), are at least 3 and less than 27 amino acids inlength, or are specific for bacteria, viruses, or cancer cells. Someantigenic domains of ligand/receptor specificity exchangers comprise apeptide having an antibody-binding region of a pathogen or toxin, are atleast 5 and less than 35 amino acids in length, or contain peptides thatare obtained from polio virus, TT virus, hepatitis B virus, and herpessimplex virus. Despite these advances in medicine, there remains a needfor more specificity exchangers that redirect antibodies present in anindividual to a target molecule.

BRIEF SUMMARY OF THE INVENTION

Aspects of the invention concern a specificity exchanger comprising aspecificity domain that is less than 200 amino acids in length joined toat least one saccharide. In some embodiments the saccharide is a Galantigen, preferably, Gal α (1,3) Gal β. These specificity exchangers canbe ligand/receptor specificity exchangers or antigen/antibodyspecificity exchangers. Although the saccharide can be directly joinedto the specificity domain such that there is no antigenic domain orlinker, some embodiments include an antigenic domain and/or linker inaddition to the saccharide.

Some embodiments of the specificity exchangers described herein bind toa bacteria (e.g., Staphylococcus), a virus (e.g., hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenzavirus, and human immunodeficiency virus (HIV)) or a cancer cell.Preferred specificity exchangers are directed to HIV and the specificitydomains of these embodiments can comprise a CD4 or CDR peptide (e.g., asequence selected from the group consisting of SEQ. ID. No. 1, SEQ. ID.No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6,SEQ. ID. No. 7, SEQ. ID. No. 8, and SEQ. ID. No. 9) and said at leastone saccharide is Gal α (1,3) Gal β. The specificity exchangersdescribed above can have a specificity domain that is less than 150,100, 50, or 25 amino acids in length. The specificity exchangersdescribed herein can be used to reduce the proliferation of bacteria,virus or cancer cells in a subject in need thereof and to preparemedicaments and pharmaceuticals for this purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method that can be employed to artificiallysynthesize glycopeptide libraries.

FIG. 2 illustrates a method to artificially synthesize cyclicglycopeptides.

FIG. 3 illustrates the different reactivity of CD4 ligand/receptorspecificity exchangers comprising glycosylated (gal-α-1,3 gal-β) orunglycosylated antigenic domains to four different human sera samples.The “X” axis shows the specificity exchangers provided at 5 μg/ml andthe “Y” axis shows the OD at 405/650 after detection of antibodiesbound.

FIG. 4 illustrates the inhibition of binding of CD4 ligand/receptorspecificity exchangers comprising glycosylated (gal-α-1,3 gal-β) orunglycosylated antigenic domains in the presence of glycosylated(gal-α-1,3 gal-β) bovine serum albumin (BSA). The “X” axis shows theconcentration of peptide and the “Y” axis shows the OD at 405/650 afterdetection of antibodies bound.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that antibody/antigen specificity exchangers andligand/receptor specificity exchangers (collectively referred to as“specificity exchangers”) that comprise saccharides or glycoconjugates(e.g., blood group sugars) react strongly to antibodies that arenaturally present in a subject and thereby promote the redirection ofsaid antibodies to a pathogen. Aspects of the invention concernspecificity exchangers (e.g., antibody/antigen specificity exchangersand ligand/receptor specificity exchangers) that comprise a saccharide,preferably a blood group sugar and more preferably a gal-α-1-3 gal βsugar. Embodiments also include pharmaceuticals comprising saidspecificity exchangers, which can be used to treat human disease, suchas infection by a pathogen or cancer. Accordingly, methods of makingsaid glycosylated specificity exchangers and using said specificityexchangers to redirect antibodies to a molecule present on a pathogen,for example, are embodiments.

Specificity exchangers comprise a specificity domain and an antigenicdomain. The length of the specificity domain of the specificityexchangers is desirably between at least 3-200 amino acids, preferablybetween at least 5-100 amino acids, more preferably between 8-50 aminoacids, and still more preferably between 10-25 amino acids. The lengthof the antigenic domain of the specificity exchangers is desirablybetween at least 3-200 amino acids, preferably between at least 5-100amino acids, more preferably between 8-50 amino acids, and still morepreferably between 10-25 amino acids. In some embodiments, however, thespecificity exchanger comprises only a glycosylated specificity domain(e.g., a portion of an antibody directed to a pathogen or a ligand for areceptor on a pathogen) such that the glycosylation region itself servesas the antigenic domain. That is, some aspects of the inventiondescribed herein concern specificity exchangers (i.e., antigen/antibodyand ligand/receptor specificity exchangers) that comprise specificitydomains directed to epitopes or receptors present on a pathogen orcancer cell, wherein said specificity domains are joined to one or moresugars (e.g., a glycosylation domain having one or more gal-α-1-3 gal βsugars) that is itself an antigenic domain that interacts withantibodies that are naturally present in a subject.

The specificity exchangers described herein comprise specificity domainsthat interact with antigens or receptors on pathogens, including, butnot limited to, bacteria, yeast, parasites, fungus, cancer cells, andpathogenic peptides. Some embodiments, for example, comprise a sequenceobtained from an antibody that binds to a bacteria, hepatitis virus(e.g., HAV, HBV, or HCV), HIV, flu viruses such as influenza virus,cancer cell epitopes, and peptides associated with human disease (e.g.,prion peptides, Alzheimer's peptides (Aβ), and neuropeptides). Otherembodiments have a specificity domain that comprises a fragment of anextracellular matrix protein (e.g., between 3 and 14 amino acids, suchas 3 to 5, 8, 9, 10, 12, or 14 consecutive amino acids of fibrinogen), aligand for a receptor on a virus (e.g., HAV, HBV, HCV, HIV, influenzavirus), or a ligand for a receptor on a cancer cell or pathogenicpeptide. In preferred embodiments, for example, the specificity domaincomprises a ligand that is a fragment (e.g., between 3 and 20 aminoacids, such as 3 to 5, 8, 9, 10, 12, 14, 17, and 20 consecutive aminoacids) of an extracellular matrix protein selected from the groupconsisting of fibrinogen, collagen, vitronectin, laminin, plasminogen,thrombospondin, and fibronectin. Several of the specificity exchangersdescribed herein bind to a receptor found on a pathogen (vis a visantigen/antibody interaction or ligand/receptor interaction). In someembodiments, the receptor is a bacterial adhesion receptor, for example,a bacterial adhesion receptor selected from the group consisting ofextracellular fibrinogen binding protein (Efb), collagen bindingprotein, vitronectin binding protein, laminin binding protein,plasminogen binding protein, thrombospondin binding protein, clumpingfactor A (ClfA), clumping factor B (ClfB), fibronectin binding protein,coagulase, and extracellular adherence protein. The next sectiondescribes specificity domains of Antigen/Antibody specificity exchangersin greater detail.

Specificity Domains of Antigen/Antibody Specificity Exchangers

The specificity domain of antigen/antibody specificity exchangers caninclude the amino-acid sequence of any antibody that specifically bindsto a certain antigen, such as a hapten, for example. Preferredspecificity domains of antigen/antibody specificity exchangers comprisean amino acid sequence of a complementarity determining region (CDR) ora framework region of a certain antibody. The CDRs of antibodies areresponsible for the specificity of the antibody. X-ray crystallographyhas shown that the three CDRs of the variable (V) region of the heavychain and the three CDRs of the V region of the light chain may all havecontact with the epitope in an antigen-antibody complex.

In certain embodiments, single peptides corresponding to the CDRs ofmabs to various antigens and that are capable of mimicking therecognition capabilities of the respective mAb can be included in thespecificity domain of the antigen/antibody specificity exchangers.Specifically, a peptide corresponding to CDRH3 of a mAb specific for theV3 region of HIV-1 gp160 or a portion of an antibody specific for aregion of gp120 that interacts with CD4 can be included in thespecificity domain. The peptide directed to the V3 region of HIV-1 wasshown to have neutralizing capacity when assayed in vitro. The CDRH3 canbe derived from mAb F58, and Ab C1-5, and the like. Like CDRH3, theCDRH1 and/or CDRH2 domain of Ab C1-5 can also be used in the specificitydomains described herein. In other embodiments the specificity domaincan include a peptide corresponding to CDRH2 of a mAb to hepatitis Bvirus core antigen (HBcAg). CDRH2 has demonstrated an ability to captureHBcAg. Several other peptides, derived from antibodies that bind HBcAgor hepatitis B virus e antigen (HBeAg) have been identified. (See U.S.Pat. No. 6,417,324, issued Jul. 9, 2002; and U.S. patent applicationSer. No. 09/839,447, filed Apr. 20, 2001 and U.S. patent applicationSer. No. 10/153,271, filed May 21, 2002, all of which are herebyincorporated by reference in their entireties). These peptides(specificity domains) can be incorporated into antigen/antibodyspecificity exchangers so as to redirect antibodies present in a subjectto hepatitis B virus. The next section describes specificity domains forligand/receptor specificity exchangers in greater detail.

Specificity Domains for Ligand/Receptor Specficity Exchangers

The diversity of ligand/receptor specificity exchangers is also equallyvast because many different ligands that bind many different receptorson many different pathogens can be incorporated into a ligand/receptorspecificity exchanger. The term “pathogen” generally refers to anyetiological agent of disease in an animal including, but not limited to,bacteria, parasites, fungus, mold, viruses, and cancer cells. Similarly,the term “receptor” is used in a general sense to refer to a molecule(usually a peptide other than a sequence found in an antibody, but canbe a carbohydrate, lipid, or nucleic acid) that interacts with a“ligand” (usually a peptide other than a sequence found in an antibody,or a carbohydrate, lipid, nucleic acid or combination thereof). Thereceptors contemplated do not have to undergo signal transduction andcan be involved in a number of molecular interactions including, but notlimited to, adhesion (e.g., integrins) and molecular signaling (e.g.,growth factor receptors).

In certain embodiments, desired specificity domains include a ligandthat has a peptide sequence that is present in an extracellular matrixprotein (e.g., fibrinogen, collagen, vitronectin, laminin, plasminogen,thrombospondin, and fibronectin) and some specificity domains comprise aligand that interacts with a bacterial adhesion receptor (e.g.,extracellular fibrinogen binding protein (Efb), collagen bindingprotein, vitronectin binding protein, laminin binding protein,plasminogen binding protein, thrombospondin binding protein, clumpingfactor A (ClfA), clumping factor B (ClfB), fibronectin binding protein,coagulase, and extracellular adherence protein).

Investigators have mapped the regions of extracellular matrix proteinsthat interact with several receptors. (See e.g., McDevvit et al., Eur.J. Biochem., 247:416-424 (1997); Flock, Molecular Med. Today, 5:532(1999); and Pei et al., Infect. and Immun. 67:4525 (1999), all of whichare herein expressly incorporated by reference in their entirety). Somereceptors bind to the same region of the extracellular matrix protein,some have overlapping binding domains, and some bind to differentregions altogether. Preferably, the ligands that make up the specificitydomain have an amino acid sequence that has been identified as beinginvolved in adhesion to an extracellular matrix protein. It should beunderstood, however, that random fragments of known ligands for anyreceptor on a pathogen can be used to generate ligand/receptorspecificity exchangers and these candidate ligand/receptor specificityexchangers can be screened in the characterization assays describedinfra to identify the molecules that interact with the receptors on thepathogen.

Some specificity domains have a ligand that interacts with a bacterialadhesion receptor including, but not limited to, extracellularfibrinogen binding protein (Efb), collagen binding protein, vitronectinbinding protein, laminin binding protein, plasminogen binding protein,thrombospondin binding protein, clumping factor A (ClfA), clumpingfactor B (ClfB), fibronectin binding protein, coagulase, andextracellular adherence protein. Ligands that have an amino acidsequence corresponding to the C-terminal portion of the gamma-chain offibrinogen have been shown to competitively inhibit binding offibrinogen to ClfA, a Staphylococcus aureus adhesion receptor. (McDevvitet al., Eur. J. Biochem., 247:416-424 (1997)). Further, Staphylococcusorganisms produce many more adhesion receptors such as Efb, which bindsto the alpha chain fibrinogen, ClfB, which interacts with both the α andβ chains of fibrinogen, and Fbe, which binds to the γ chain offibrinogen. (Pei et al., Infect. and Immun. 67:4525 (1999)).Accordingly, preferred specificity domains comprise between 3 and 30amino acids, that is, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30consecutive amino acids of a sequence present in a molecule (e.g.,fibrinogen) that can bind to a bacterial adhesion receptor.

Specificity domains can also comprise a ligand that interacts with aviral receptor. Several viral receptors and corresponding ligands areknown and these ligands or fragments thereof can be incorporated into aligand/receptor specificity exchanger. For example, Tong et al., hasidentified an Hepadnavirus receptor, a 170 kd cell surface glycoproteinthat interacts with the pre-S domain of the duck hepatitis B virusenvelope protein (U.S. Pat. No. 5,929,220) and Maddon et al., hasdetermined that the T cell surface protein CD4 (or the soluble formtermed T4) interacts with gp120 of HIV (U.S. Pat. No. 6,093,539); bothreferences are herein expressly incorporated by reference in theirentireties. Thus, specificity domains that interact with a viralreceptor can comprise regions of the pre-S domain of the duck hepatitisB virus envelope protein (e.g., amino acid residues 80-102 or 80-104) orregions of the T cell surface protein CD4 (or the soluble form termedT4) that interacts with gp120 of HIV (e.g., the extracellular domain ofCD4/T4 or fragments thereof). For example, ligand/receptor specificitydomains directed to the CDR (V3 binding complement) or CD4 (gp120binding complement) binding domains of HIV have been prepared. (SeeTABLE 1). Many more ligands for viral receptors exist and thesemolecules or fragments thereof can be used as a specificity domain.

TABLE 1 HIV specific ligand/receptor specificity domains GlycosylatedSp.exchanger Name Sequence Identifier Gal-CDR DCDLIYYDYEEDYYFDY (Seq.Id. No. 1) Gal-CD4-1 DQFHWKNSNQIKILGN (Seq. Id. No. 2) Gal-CD4-4DQGSFLTKGPSKLNDR (Seq. Id. No. 3) Gal-CD4-100 QFHWKNSNQIKILGN (Seq. Id.No. 4) Gal-CD4-101 NSNQIKILGNQGSFL (Seq. Id. No. 5) Gal-CD4-102KILGNQGSFLTKGPS (Seq. Id. No. 6) Gal-CD4-103 QGSFLTKGPSKLNDR (Seq. Id.No. 7) Gal-CD4-104 TKGPSKLNDRADSRR (Seq. Id. No. 8) Gal-CD4-105KLNDRADSRRSLWDQ (Seq. Id. No. 9)

Specificity domains can also comprise a ligand that interacts with areceptor present on a cancer cell. The proto-oncogene HER-2/neu(C-erbB2) encodes a surface growth factor receptor of the tyrosinekinase family, p185HER2. Twenty to thirty percent of breast cancerpatients over express the gene encoding HER-2/neu (C-erbB2), via geneamplification. Thus, ligand/receptor specificity exchangers comprising aspecificity domain that encodes a ligand for HER-2/neu (C-erbB2) aredesirable embodiments. Many types of cancer cells also over express ordifferentially express integrin receptors. Many preferred embodimentscomprise a specificity domain that interacts with an integrin receptor.Although integrins predominantly interact with extracellular matrixproteins, it is known that these receptors interact with other ligandssuch as invasins, RGD-containing peptides (i.e.,Arginine-Glycine-Aspartate), and chemicals. (See e.g., U.S. Pat. Nos.6,090,944 and 6,090,388; and Brett et al., Eur J Immunol, 23:1608(1993), all of which are hereby expressly incorporated by reference intheir entireties). Ligands for integrin receptors include, but are notlimited to, molecules that interact with a vitronectin receptor, alaminin receptor, a fibronectin receptor, a collagen receptor, afibrinogen receptor, an integrin receptor. The next section describessome of the antigenic domains that can be used with the specificityexchangers described herein.

Antigenic Domains

The diversity of antigenic domains that can be used in theligand/receptor specificity exchangers and antibody/antigen specificityexchangers is quite large because a pathogen or toxin can present manydifferent epitopes. Desirably, the antigenic domains used with thespecificity exchangers are peptides obtained from surface proteins orexposed proteins from bacteria, fungi, plants, molds, viruses, cancercells, and toxins. It is also desired that the antigenic domainscomprise a peptide sequence that is rapidly recognized as non-self byexisting antibodies in a subject, preferably by virtue of naturallyacquired immunity or vaccination. For example, many people are immunizedagainst childhood diseases including, but not limited to, small pox,measles, mumps, rubella, and polio. Thus, antibodies to epitopes onthese pathogens can be produced by an immunized person. Desirableantigenic domains have a peptide that contains one or more epitopes thatis recognized by antibodies in the subject that are present in thesubject to respond to pathogens such as small pox, measles, mumps,rubella, herpes, hepatitis, and polio.

Some embodiments, however, have antigenic domains that interact with anantibody that has been administered to the subject. For example, anantibody that interacts with an antigenic domain on a specificityexchanger can be co-administered with the specificity exchanger.Further, an antibody that interacts with a specificity exchanger may notnormally exist in a subject but the subject has acquired the antibody byintroduction of a biologic material or antigen (e.g., serum, blood, ortissue) so as to generate a high titer of antibodies in the subject. Forexample, subjects that undergo blood transfusion acquire numerousantibodies, some of which can interact with an antigenic domain of aspecificity exchanger. Some preferred antigenic domains for use in aspecificity exchanger also comprise viral epitopes or peptides obtainedfrom pathogens such as the herpes simplex virus, hepatitis B virus, TTvirus, and the poliovirus.

Preferably, the antigenic domains comprise an epitope or peptideobtained from a pathogen or toxin that is recognized by a “high-titerantibody.” The term “high-titer antibody” as used herein, refers to anantibody that has high affinity for an antigen (e.g., an epitope on anantigenic domain). For example, in a solid-phase enzyme linkedimmunosorbent assay (ELISA), a high titer antibody corresponds to anantibody present in a serum sample that remains positive in the assayafter a dilution of the serum to approximately the range of 1:100-1:1000in an appropriate dilution buffer. Other dilution ranges include1:200-1:1000, 1:200-1:900, 1:300-1:900, 1:300-1:800, 1:400-1:800,1:400-1:700, 1:400-1:600, and the like. In certain embodiments, theratio between the serum and dilution buffer is approximately: 1:100,1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600,1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000. Epitopes orpeptides of a pathogen that can be included in an antigenic domain of aspecificity exchanger include the epitopes or peptide sequencesdisclosed in Swedish Pat No. 9901601-6; U.S. Pat. No. 5,869,232; Mol.Immunol. 28: 719-726 (1991); and J. Med. Virol. 33:248-252 (1991); allwhich are herein expressly incorporated by reference in theirentireties.

The antigenic domains of the specificity exchangers described herein donot have to be peptides, however. In some embodiments, the sugar,plurality of sugars, glycosylation region or glycosylation domain isitself the antigenic domain. That is, some embodiments are specificityexchangers (i.e., antigen/antibody and ligand/receptor specificityexchangers) that comprise a specificity domain that is joined to asugar, a plurality of sugars, a glycosylation region, or a glycosylationdomain with or without a peptide linker but lacking an antigenic peptideor epitope obtained from a pathogen or toxin. In this manner,glycosylated specificity domains (e.g., antigen/antibody andligand/receptor specificity domains) are also referred to asglycosylated specificity exchangers, wherein the sugar, plurality ofsugars, glycosylation region or glycosylation domain is itself theantigenic domain. The next section describes glycosylated specificityexchangers in greater detail.

Specificity Exchangers Comprising Saccharides and Glycoconjugates

Generally, the glycosylated specificity exchangers (i.e.,antibody/antigen specificity exchangers and ligand/receptor specificityexchangers) comprise a specificity domain that is at least 3 and lessthan or equal to 200 amino acids in length joined to an antigenic domain(e.g., a peptide backbone) that is at least 3 and less than or equal to200 amino acids in length or no peptide-based antigenic domain at all(i.e., the specificity domain is glycosylated itself with or without alinker but lacking an antigenic peptide obtained from a pathogen orcontaining an epitope of a pathogen). The antigenic domain and/orspecificity domain can comprise a plurality of saccharides that,together with the peptide backbone or by itself, react with high titerantibodies that are naturally present in a human. Preferably, theglycosylation domain or region contains blood group sugars that arexenoactive antigens (e.g., blood group sugars that are the basis forhyperactute rejection of xenografts or transplantations).

In some embodiments, for example, the specificity exchangers comprise aspecificity domain that is between or at least and/or less than or equalto 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 amino acids in length, and said specificity domain is joinedto an antigenic domain (e.g., a peptide backbone) that is between or atleast and/or less than or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length, whereinsaid antigenic domain or specificity domain or both comprise a pluralityof saccharides. Other embodiments comprise a specificity domain that isbetween or at least and/or less than or equal to 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids inlength, and said specificity domain is joined to a plurality ofsaccharides (with or without a peptide linker and with or without apeptide or epitope of a pathogen or with or without an antigenicdomain). Depending on the embodiment, the “plurality of saccharides” caninclude at least 2 and 10,000 or more sugar units. In some embodiments,for example, between or at least and/or less than or equal to 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750,4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 7000, 8000, 9000,10,000 or more sugar units are joined to the specificity domain eitherdirectly or indirectly (e.g., through a support such as the peptidebackbone of a linker an antigenic domain comprising a peptide or epitopeof a pathogen).

The diversity of specificity domains that can be used in the specificityexchangers described herein is quite large because many differentantibody/antigen and ligand/receptor interactions exist on a pathogen(e.g., a bacteria such as Staphylococcus). Preferred specificity domainsare directed to bacterial adhesion proteins such as ClfA and ClfB orother bacterial receptors that interact with fragments of fibrinogen andspecificity domains directed to viruses such as hepatitis, flu, and HIV.The diversity of antigenic domains that can be used in the specificityexchangers described herein is also quite large because many differentsupports and many different saccharides or groups of saccharides can beused. The term “saccharide” is intended to be construed broadly so as tonon-exclusively encompass monosaccharides, disaccharides,polysaccharides (glycans), oligosaccharides, and other similarcompounds. The term “glycoconjugate” is also to be construed broadly,and generally refers to an organic compound consisting of one or morecarbohydrate units (e.g., a saccharide) joined to a support.

In several embodiments, the specificity domain is joined to a support towhich a plurality of saccharides and/or a glycoconjugate is also joined.A “support” can be a peptide backbone, (e.g., an antigenic domain, asdescribed above), a protein, a resin, or any macromolecular structurethat can be used to join or immobilize a saccharide or a specificitydomain. The saccharides and specificity domains can be joined toinorganic supports, such as silicon oxide material (e.g., silica gel,zeolite, diatomaceous earth or aminated glass) by, for example, acovalent linkage through a hydroxy, carboxy, or amino group and areactive group on the support. In some embodiments, the support has ahydrophobic surface that interacts with a portion of the specificitydomain and/or saccharide or saccharide conjugate (e.g., glycolipid) by ahydrophobic non-covalent interaction. In some cases, the hydrophobicsurface of the support is a polymer such as plastic or any other polymerin which hydrophobic groups have been linked such as polystyrene,polyethylene or polyvinyl.

Additionally, supports such as proteins and oligo/polysaccarides (e.g.,cellulose, starch, glycogen, chitosane or aminated sepharose) can beused by exploiting reactive groups on the specificity domains orsaccharides, such as a hydroxy or an amino group, to join to a reactivegroup on the support so as to create the covalent bond. Still moresupports containing other reactive groups that are chemically activatedso as to attach the saccharides and specificity domains can be used(e.g., cyanogen bromide activated matrices, epoxy activated matrices,thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxysuccinimide chlorformate linkages, or oxirane acrylic supports).

The insertion of linkers (e.g., “λ linkers” engineered to resemble theflexible regions of λ phage) of an appropriate length between thespecificity domain and/or the plurality of saccharides and the supportare also contemplated so as to encourage greater flexibility andovercome any steric hindrance that can be encountered. The determinationof an appropriate length of linker that allows for optimal binding canbe found by screening the attached molecule with varying linkers in thecharacterization assays detailed herein.

Preferred embodiments include specificity exchangers that compriseglycoconjugates and support-bound saccharides that are commonly referredto as glycoproteins, proteoglycans, glycopeptides, peptidoglycans,glyco-amino-acids, glycosyl-amino-acids, glycolipids, and relatedcompounds. The glycoproteins that can be used with an embodimentdescribed herein include compounds that contain a carbohydrate and aprotein. The carbohydrate may be a monosaccharide, disaccharide(s),oligosaccharide(s), polysaccharide(s), their derivatives (e.g., sulfo-or phospho-substituted), and other similar compounds. There are twomajor classes of glycoproteins that can be used, O-linked glycans andthe N-linked glycans. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The most common O-linkage involves aterminal N-acetylgalactosamine residue in the oligosaccharide linked toa serine or threonine residue of the protein. While specificityexchangers that comprise a glycoprotein can include one, a few, or manycarbohydrate units, some embodiments comprise a proteoglycan, a subclassof glycoproteins that are polysaccharides that contain amino sugars.

The glycopeptides that can be used with some of the embodimentsdescribed herein include compounds having a carbohydrate linked to anoligopeptide composed of L- and/or D-amino acids. The peptidoglycansthat can be used comprise a glycosaminoglycan formed by alternatingresidues of D-glucosamine and either muramic acid{2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose} orL-talosaminuronic acid (2-amino-2-deoxy-L-taluronic acid), which areusually N-acetylated or N-glycosylated.

The glyco-amino-acids that can be used with the embodiments describedherein comprise a saccharide attached to a single amino acid, whereasthe glycosyl-amino-acids that can be used include compounds comprising asaccharide linked through a glycosyl linkage (O—, N— or S—) to an aminoacid. (The hyphens are used to avoid implying that the carbohydrate isnecessarily linked to the amino group.) In some embodiments, theantigenic domain comprises a glycolipid, which is a compound comprisingone or more monosaccharide residues bound by a glycosidic linkage to ahydrophobic moiety such as an acylglycerol, a sphingoid, a ceramide(N-acylsphingoid) or a prenyl phosphate, for example. Some of thespecificity exchangers described herein can also comprise aglycoconjugate (e.g., lectins).

Preferred embodiments, however, include specificity exchangers thatcomprise human proteins or glycoconjugates that are commonly referred toas blood group antigens. These antigens are generally surface markerslocated on the outside of red blood cell membranes. Most of thesesurface markers are proteins, however, some are carbohydrates attachedto lipids or proteins. Structurally, the blood group determinants thatcan be used with the embodiments described herein fall into two basiccategories known as type I and type II. Type I comprises a backbonecomprised of a galactose 1-3β linked to N-acetyl glucosamine while typeII comprises, instead, a 1-4 β linkage between the same building blocks(cf. N-acetyl lactosamine). The position and extent of a-fucosylation ofthese backbone structures gives rise to the Lewis-type and H-typespecificities. Thus, monofucosylation at the C₄-hydroxyl of the N-acetylglucosamine (Type I series) constitutes the Le^(a) type, whereasfucosylation of the C₃-hydroxyl of this sugar (Type II series)constitutes the Le^(x) determinant. Additional fucosylation of Le^(a)and Le^(x) types at the C_(2,)-hydroxyl of the galactose sectorspecifies the Le^(b) and Le^(y) types, respectively.

The presence of an a-monofucosyl branch, solely at the C_(2,)-hydroxylin the galactose moiety in the backbone, constitutes the H-typespecifity (Types I and II). Further permutation of the H-types bysubstitution of a-linked galactose or a-linked N-acetylgalactosamine atits, -hydroxyl group provides the molecular basis of the familiarserological blood group classifications A, B, and O. (See e.g., Lowe, J.B., The Molecular Basis of Blood Diseases, Stamatoyannopoulos, et. al.,eds., W.B. Saunders Co., Philadelphia, Pa., 1994, 293., herein expresslyincorporated by reference in its entirety.)

By first determining a patient's particular set of blood group antigens,one can select a specificity exchanger comprising one or more bloodgroup antigens that are outside of the repertoire of the patient so asto generate a potent response to the antigenic domain of the specificityexchanger in the patient and thereby redirect the antibodies present inthe patient to the pathogen that is specific for the specificity domainof the specificity exchanger. Accordingly, specificity exchangers thatare specific for several different pathogens can be made to haveantigenic domains that comprise many different combinations of bloodgroup antigens so that a potent immune response can be obtained in anyparticular individual. The next section describes the manufacture ofspecificity exchangers comprising saccharides and glycoconjugates, inparticular blood group antigens, in greater detail.

Making Specificity Exchangers that Comprise Saccharides andGlycoconjugates

The manufacture of antigen/antibody specificity exchangers andligand/receptor specificity exchangers has been described previously.(See e.g., U.S. Pat. Nos. 5,869,232; 6,040,137; 6,245,895; 6,417,324;6,469,143; 6,660,842; and U.S. application Ser. Nos. 09/839,447;09/839,666; 09/664,945; 10/372,735; and 09/664,025; and InternationalApp. Nos. PCT/SE95/00468 and PCT/IB01/00844, and PCT/IB01/02327, all ofwhich are herein expressly incorporated by reference in theirentirities). The manufacture of these specificity exchangers can bemodified so as to join or incorporate saccharides and glycoconjugatesaccording to methods that are known in the art.

Several issues merit consideration in contemplating the synthesis ofsuch blood group substances and their neoglycoconjugates, however. Forpurposes of synthetic economy it is helpful to gain relief fromelaborate protecting group manipulations common to traditional synthesesof complex branched carbohydrates. Another issue involves fashioning adeterminant linked to a protein carrier. In crafting such constructs, itmay be beneficial to incorporate appropriate spacer units between thecarbohydrate determinant and the carrier. (See e.g., Stroud, M. R., etal., Biochemistry, 1994, 33, 10672; Yuen, C.-T., et al., J. Biochem.,1994, 269, 1595; and Stroud, M. R., et al., J. Biol. Chem., 1991, 266,8439, all of which are herein expressly incorporated by reference inentirities)

Table 2 provides a non-exclusive list of blood group antigens that canbe joined to or incorporated in a specificity exchanger.

TABLE 2 Blood group carrier or SWISS-PROT effector protein name crossAntigen system Gene name reference names ABO Fucosylglycoproteinalpha-n- Gene: ABO BGAT_HUMAN Antigens: A/Bacetylgalactosaminyltransferase (P16442) (EC 2.4.1.40) (Histo- bloodgroup A transferase)/ Fucosylglycoprotein 3-alpha- galactosyltransferase(EC 2.4.1.37) (Histo-blood group B transferase) Chido/ Complement C4Gene: C4A and C4B C04_HUMAN Antigens: Ch1 Rodgers (P01028) to Ch6, WH,Rg1, Rg2 Colton Aquaporin-CHIP (Aquaporin Gene: AQP1; AQP1_HUMANAntigens: 1). CHIP28 (P29972) Co(a/b) Cromer Complement decay- Gene:DAF; CD55 DAF_HUMAN Antigens: accelerating factor (Antigen (P08174)Cr(a), Dr(a), CD55). Es(a), Tc(a/b/c), Wd(a), WES(a/b), IFC, UMC DiegoBand 3 anion transport Gene: SLC4A1; B3AT_HUMAN Antigens: protein (Anionexchange AE1; EPB3 (P02730) Di(a/b), protein 1) (AE 1). Wr(a/b), Wd(a),Rb(a), WARR Dombrock Dombrock glycoprotein. Gene: DO Not yet Antigens:identified Do(a/b), Gy(a), Hy, Jo(A) Duffy Duffy antigen (Fy Gene: FY;GPD; DUFF_HUMAN Antigens: glycoprotein) (Glycoprotein DARC (Q16570)Fy(a/b) D) (GpFy). Gerbich Glycophorin C (PAS-2′) Gene: GYPC; GPCGLPC_HUMAN Antigens: (Glycoprotein beta) (P04921) An(a), Dh(A),(Glycoconnectin) Ls(a), Wb (Sialoglycoprotein D) (Glycophorin D) (GpD).Hh Galactoside 2-L- Gene: FUT1 FUT1_HUMAN fucosyltransferase 1 (EC(P19526) 2.4.1.69) (Alpha(1,2)Ft 1) (Fucosyltransferase 1). Galactoside2-L- Gene: FUT2 FUT2_HUMAN Antigens: H/h, fucosyltransferase 2 (EC(Q10981) Se/se 2.4.1.69) (Alpha(1,2)Ft 2) (Fucosyltransferase 2)(Secretor factor). Indian CD44 antigen (Phagocytic Gene: CD44; LHRCD44_HUMAN Antigens: glycoprotein I) (PGP-1) (P16070) In(a/b) (Hutch-I)(Extracellular matrix receptor-III) (ECMR- III) (Hermes antigen)(Hyaluronate receptor) (Heparan sulfate proteoglycan) (Epican). KellKell blood group glycoprotein Gene: KEL KELL_HUMAN Antigens: K/k, (EC3.4.24.-). (P23276) Kp(a/b/c), Js(a/b), Ul(a), KEL11/17, KEL14/24 KiddUrea transporter, Gene: SLC14A1; UT1_HUMAN Antigens: erythrocyte. UT1;HUT11; UTE; (Q13336) Jk(a/b) JK; RACH1 Knops Complement receptor type 1Gene: CR1; C3BR CR1_HUMAN Antigens: (C3b/C4b receptor) (Antigen (P17927)Kn(a/b), CD35). McC(a), Sl(a), Yk(a) Kx Membrane transport protein Gene:XK XK_HUMAN XK (Kx antigen). (P51811) Landsteiner- Landsteiner-Wienerblood Gene: LW LW_HUMAN Antigens: Wiener group glycoprotein. (Q14773)Lw(a/b) Lewis Galactoside 3(4)-L- Gene: FUT3; LE FUT3_HUMAN Antigens:fucosyltransferase (EC (P21217) Le(a/b) 2.4.1.65) (Fucosyltransferase 3)(FUCT-III). Lutheran Lutheran blood group Gene: LU; BCAM; LU_HUMANAntigens: glycoprotein (B-CAM cell MSK19 (P50895) Lu(a/b), surfaceglycoprotein) Au(a/b), LU6 (Auberger B antigen) to LU20 (F8/G253antigen). MNS Glycophorin A (PAS-2) Gene: GYPA; GPA GLPA_HUMAN Antigens:M/N, (Sialoglycoprotein alpha) (P02724) S/s, U, He, (MNsialoglycoprotein). Mi(a), M(c), Glycophorin B (PAS-3) Gene: GYPB; GPBGLPB_HUMAN Vw, Mur, M(g), (Sialoglycoprotein delta) (P06028) Vr, M(e),(SS-active sialoglycoprotein). Mt(a), St(a), Ri(a), Cl(a), Ny(a), Hut,Hil, M(v), Far, Mit, Dantu, Hop, Nob, En(a), ENKT, etc. P A yetundefined Gene: P1 Not yet Antigens: P1 galactoyltransferase. identifiedRh Blood group RH(CE) Gene: RHCE; RHC; RHCE_HUMAN Antigens: C/c,polypeptide (Rhesus C/E RHE (P18577) E/e, D, f, C(e), antigens) (RHPI).C(w), C(x), V, Blood group RH(D) Gene: RHD RHD_HUMAN E(w), G, Tar,polypeptide (Rhesus D (Q02161) VS, D(W), cE, antigen) (RHPII). etc.Scianna Scianna glycoprotein. Gene: SA Not yet Antigens: identifiedSc(1/2), Sc3 Xg Xg glycoprotein (Protein Gene: XG; PBDX XG_HUMANAntigens: PBDX). (P55808) Xg(a) Yt Acetylcholinesterase (EC Gene: ACHEACES_HUMAN Antigens: 3.1.1.7). (P22303) Yt(a/b)

Additional blood groups can include Lewis^(x)-BSA, 2′-Fucosyllactose-BSA(2′FL-BSA), Lacto-N-fucopentaose II-BSA, Lacto-N-fucopentaose III-BSA,Lacto-N-fucopentaose I-BSA (LNFPI-BSA), Lacto-N-difucohexaose I-BSA(LNDFHI-BSA), Blood Group A-BSA, Blood Group B-BSA, Globotriose-HSA,Gala1-4Galb1-4Glc-HSA, and the like.

While blood group antigens have been discussed in detail, it isimportant to point out that any saccharide or glycoconjugate can beincluded in the antigenic domain of the specificity exchangers describedherein. Antigenic saccharides and glycoconjugates are well known in theart and are readily available from a commercial supplier such as V-Labs,Inc. (Covington, La.). Saccharides and glycoconjugates can also besynthesized using conventional techniques (as will be described in moredetail). Potential saccharides and glycoconjugates that can be usedherein can be derived from pathogens, including bacteria, viruses (e.g.,L, M, and S glycoproteins from HBV, and gp160, gp120 and gp41 from HIV),protozoan, and fungi, cancer cells, toxins, cells affected by autoimmunediseases such as lupus, multiple sclerosis, rheumatoid arthritis,diabetes, psoriasis, Graves disease and the like.

Specific core structure neoglycoproteins that can be used in theantigenic domains described herein include: N-Acetyllactosamine-BSA(3-atom spacer), N-Acetyllactosamine-BSA (14-atom spacer), α1-3,α1-6Mannotriose-BSA (14-atom spacer) and the like. Monosaccharideneoglycoproteins that can be used in the antigenic domains describedherein include: N-Acetylglucosamine-BSA (14-atom spacer),N-Acetylgalactosamine-BSA (14-atom spacer), and the like. Tumor antigenneoglycoproteins that can be used in the antigenic domains describedherein include: T-Antigen-HSA Galβ1-3GalNAc-HSA (3-atom spacer),Tn-Antigen-HSA GalNAca1-O-(Ser-N-Ac-CO)-Spacer-NH-HSA, and the like.Sialyated neoglycoproteins that can be used in the antigenic domainsdescribed herein include: 3′ Sialyl-N-acetyllactosamine-BSA (3-atomspacer), 3′-Sialyl-N-acetyllactosamine-BSA (14-atom spacer), 3′-SialylLewis^(x)-BSA (3-atom spacer), 3′-Sialyl Lewis^(x)-HSA (3-atom spacer),3′-Sialyl-3-Fucosyl-Lactose-BSA (3-atom spacer), 3′-Sialyl Lewis^(x)-BSA(14-atom spacer), and the like.

In certain embodiments, the antigenic domain can include Gal α (1,3) Galβ(gal antigen), a carbohydrate antigen. The gal antigen is produced inlarge amounts on the cells of pigs, mice and New World monkeys by theglycosylation enzyme galactosyltransferase (α(1,3)GT).Galactosyltransferase is active in the Golgi apparatus of cells andtransfers galactose from the sugar-donor uridine diphosphate galactose(UDP-galactose) to the acceptor N-acetyllactosamine residue oncarbohydrate chains of glycolipids and glycoproteins, to form galantigen.

The gal antigen is completely absent in humans, apes and Old Worldmonkeys because their genes encoding α (1,3) GT have become inactivatedin the course of evolution. (Xing et al., 01-2-x1 Cell Research 11(2):116-124 (2001), herein expressly incorporated by reference in itsentirety.) Since humans and Old World primates lack the gal antigen,they are not immunotolerant to it and produce anti-gal antigenantibodies (anti-Gal) throughout life in response to antigenicstimulation by gastrointestinal bacteria. (Id.) It has been estimatedthat as many as 1% of circulating B cells are capable of producing theseantibodies. (Id.) The binding of anti-Gal to gal antigens expressed onglycolipids and glycoproteins on the surface of endothelial cells indonor organs leads to activation of the complement cascade andhyperacute rejection, and also plays an important role in occurrence ofcomplement-independent delayed xenograft rejection. (Id.) Accordingly,the gal antigen has the ability to generate a potent immune response.

In certain embodiments the gal antigen to be joined or incorporated intoa specificity exchanger is selected from gal α (1,3) gal seriesneoglycoproteins and can include: Gala1-3Gal-BSA (3-atom spacer),Gala1-3Gal-BSA (14-atom spacer), Gala1-3Gal-HSA (3-atom spacer),Gala1-3Gal-HSA (14-atom spacer), Gala1-3Galβ1-4GlcNAc-BSA (3-atomspacer), Gala1-3Galp1-4GlcNAc-BSA (14-atom spacer),Gala1-3Galβ1-4GlcNAc-HSA (3-atom spacer), Gala1-3Galβ1-4GlcNAc-HSA(14-atom spacer), Galili Pentasaccharide-BSA (3-atom spacer), and thelike. In other embodiments the gal antigen can be selected from galα(1,3) gal analogue neoglycoproteins, including Gala1-3Galβ1-4Glc-BSA(3-atom spacer), Gala1-3Galβ1-4Glc-HSA (3-atom spacer),Gala1-3Galp1-3GlcNAc-BSA (3-atom spacer), Gala1-3Galβ1-3GlcNAc-HSA(3-atom spacer), Gala1-3Galβ1-4(3-deoxyGlcNAc)-HSA (3-atom spacer),Gala1-3Galβ1-4(6-deoxyGlcNAc)-HSA, and the like.

Danishefsky, et al., discloses several antigenic saccharides andglycoconjugates, and methods of synthesizing said compounds. (See U.S.Pat. No. 6,303,120, herein expressly incorporated by reference in itsentirety). Specifically, this patent provides a method of synthesizingLe^(y)-related antigens as well as artificial protein-conjugates of theoligosaccharide. In certain embodiments, these antigens contain a novelarray of features including the α-linkage between the B and the Centities, as well as the β-linked ring D gal-NAc residue. (For thesynthesis of a related structure (SSEA-3), which lacks the fucoseresidue see: Nunomura, S.; Ogawa, T., Tetrahedron Lett., 1988, 29,5681-5684., herein expressly incorporated by reference in its entirety.)In general, the methods described in U.S. Pat. No. 6,303,120, hereinexpressly incorporated by reference in its entirety can be used ormodified so as to join or incorporate the saccharides or glycoconjugatesdescribed herein with a specificity exchanger.

A major obstacle in the field of glycobiology is access to pure,chemically well defined complex carbohydrates and glycoconjugates. (SeeRandell, Karla D., et al., High-throughput Chemistry toward ComplexCarbohydrates and Carbohydrate-like Compounds, National Research Councilof Canada, publication no. 43876, Feb. 13, 2001, herein expresslyincorporated by reference in its entirety). Unlike nucleic acids andpolypeptides, these are non-linear molecules and the carbohydratemoieties present tremendous challenges in developing their totalsyntheses. (Id.) These polyhydroxy compounds contain an array ofmonosaccharide units and have a variety of glycosidic linkages betweenthem. (Id.) Each glycosidic linkage can exist in the α- or β-anomericconfiguration. (Id.) Therefore, carbohydrate syntheses can require manyorthogonal protection-deprotection schemes and involve difficultglycosyl coupling reactions. (Id.) Recently, efforts have been made todevelop automated syntheses of complex carbohydrates. (Id.)

While vastly more complicated than the techniques for synthesizingpolynucleotides and polypeptides, techniques for synthesizingsaccharides and glycoconjugates are known in the art. These techniquesare discussed in the sections that follow as they fall into enzyme-basedapproaches, cell-based approaches, and chemical synthesis-basedapproaches.

Enzyme Synthesis

Different methods for synthesizing saccharides and glycoconjugatesdescribed herein can be found in U.S. Pat. No. 6,046,040, issued toNishiguchi et al. (2000), which is hereby expressly incorporated byreference in its entirety. Specifically this patent discloses usingenzyme-catalyzed in vitro reactions to synthesize saccharides andglycoconjugates. See also Toone et al., Tetrahedron Reports (1990)(45)17:5365-5422. Enzymatic approaches have been gaining popularity forthe synthesis of saccharides and glycoconjugates in part because enzymesfeature exquisite stereo- and regioselectivity and catalyze the reactionunder very mild conditions. Extensive protection-deprotection schemesare thus unnecessary, and the control of anomeric configuration issimplified.

To produce some of the specificity exchangers described herein, thefollowing enzymes may be used: saccarglycosyltransferases, glycosidases,glycosyl hydrolases or glycosyltransferases. Glycosyltransferasesregulate the biosynthesis of carbohydrate antigens in cells and areresponsible for the addition of carbohydrates to the oligosaccharidechain on glycolipids and glycoproteins in a sequential manner.Glycosyltransferases catalyze the addition of activated sugars, in astepwise fashion, to a protein or lipid or to the non-reducing end of agrowing oligosaccharide. Typically a relatively large number ofglycosyltransferases are used to synthesize carbohydrates. EachNDP-sugar residue requires a distinct class of glycosyltransferase andeach of the more than one hundred glycosyltransferases identified todate appears to catalyze the formation of a unique glycosidic linkage.

According to one enzyme-catalsyed method of synthesis, saccharides aresynthesized using a solid phase method that utilizes glycal (Danishefskyet al., Science, 260, 1307 (1993)). This method includes (i) binding aglycal to a polystyrene-divinylbenzene copolymer via a diphenylsilylgroup to allow reaction between the glycal and 3,3-dimethyldioxirane,that converts glycal to a 1,2-anhydrosugar, and (ii) using thisanhydrosugar as a sugar donor, reaction with a different glycal suitablyprotected to form a glycoside glycal, and these steps are repeated.According to this method, a new glycosidic linkage is stereoselectivelyformed.

A solid phase method of sugar chain synthesis can also be used togenerate saccharides or glycoconjugates to be used in the specificityexchangers described herein. This method utilizes glycosyltransferase,which is capable of stereoselectively forming a glycosidic linkagewithout any protection. In the past, this method has not reached itspotential due to the fact that available glycosyltransferase is limitedin kind and is expensive. In recent years, however, genes of variousglycosyltransferases have been isolated and a large-scale production ofglycosyltransferase by genetic techniques is common place.

U. Zehavi et al. reports a solid phase synthesis method that can be usedto manufacture some of the specificity exchangers described herein,whereby a glycosyltransferase and a polyacrylamide gel bound with anaminohexyl group on a solid phase carrier is used. (See Carbohydr. Res.,124, 23 (1983), Carbohydr. Res., 228, 255 (1992), hereby expresslyincorporated by reference in its entirety). This method comprises thesteps of converting a suitable monosaccharide to4-carboxy-2-nitrobenzylglycoside, condensing this glycoside with theamino group of the above-mentioned carrier, elongating the sugar chainby glycosyltransferase using the condensate as a primer, and releasingthe oligosaccharide by photolysis.

In the past, there was a common understanding that glycosyltransferasedoes not react well with saccharide or oligosaccharide bound to a solidphase carrier, and that efficient elongation of a sugar chain isdifficult to achieve. However, more recently it has been discovered thatthe linkage between 4-carboxy-2-nitrobenzylglycoside and solid phasecarrier by a linker having a long chain, such as hexamethylene andoctamethylene, improved sugar transfer yield at the maximum of 51%(React. Polym., 22, 171 (1994), Carbohydr. Res., 265, 161 (1994)).

C. H. Wong et al. report a method of enzymatic synthesis wherebyglycosyltransferase is used to elongate sugar residues bound to aminatedsilica and, once complete, the elongated sugar chain is cleaved from thesupport using α-chymotrypsin. (See J. Am. Chem. Soc., 116, 1136 (1994),which is hereby expressly incorporated by reference in its entirety). Bythis method, the transglycosylation yield was 55%. Similarly, M. Meldalet al. reports another method of elongating a sugar chain usingglycosyltransferase and a polymer of mono- and diacryloyl compound ofdiaminated poly(ethylene glycol) as a primer. The sugar chain wasreleased by trifluoroacetic acid. (See J. Chem. Soc., Chem. Commun.,1849 (1994), which is hereby expressly incorporated by reference in itsentirety). As mentioned above, when a sugar chain is elongated byglycosyltransferase on a solid phase carrier, the kind of group (linker)that connects the solid phase carrier to the sugar residue (receptor ofinitial transglycosylation) varies transglycosylation yield. When thesugar chain is liberated from the carrier, the presence of aspecifically cleavable bond in the linker is desired. In sugar chainelongation by glycosyltransferase, the use of an immobilizedglycosyltransferase that permits repetitive use is also desired.Preferably, if an immobilized glycosyltransferase is used for sugarchain elongation, the reaction is carried out on a water solublecarrier.

U.S. Pat. No. 6,046,040, issued to Nishiguchi et al. (2000), which ishereby expressly incorporated by reference in its entirety, describessugar chain synthesis using an immobilized glycosyltransferase and awater soluble carrier. Accordingly, by one approach to generate thesugar-containing antigenic domains described herein, the following stepscan be employed: (i) binding a sugar residue to the side chain of awater-soluble polymer via a linker having a selectively cleavablelinkage to give a primer, and bringing said primer into contact with animmobilized glycosyltransferase in the presence of a sugar nucleotide,to transfer a sugar residue of said sugar nucleotide to the sugarresidue of said primer, (ii) elongating a sugar chain by transfer ofplural sugar residues by repeating the step (i) at least once, (iii)removing, where necessary, a by-produced nucleotide or an unreactedsugar nucleotide, and (iv) repeating the steps (i)-(iii) where necessaryand releasing the sugar chain by selectively cleaving the cleavablelinkage in the linker, from the above-mentioned primer connecting thesugar chain elongated by the transfer of plural sugar residues. Themethods disclosed in U.S. Pat. No. 6,046,040 can be used to synthesizeglycoconjugates having an optional sugar chain structure, such asoligosaccharides, glycopeptides and glycolipids, as well. Theapplication of enzymes to an automated scheme of saccharide orglycoconjugate synthesis is also possible. Both solution and solid-phasemethods can be used for automated synthesis.

In some embodiments, an apparatus that utilize enzymes to synthesizesaccharides and glycoconjugates can be used herein. U.S. Pat. No.5,583,042, which is hereby expressly incorporated by reference in itsentirety, for example, describes an apparatus that utilizes combinationsof glycosyltransferases, for the synthesis of specific saccharides andglycoconjugates. The next section describes several cell-basedapproaches to manufacture specificity exchangers comprising saccharidesor glycoconjugates.

Cell Based Synthesis

In addition to using in vitro enzyme catalyzed reactions, any availablecell-based methods can be used to synthesize the saccharides andglycoconjugates described herein. U.S. Pat. No. 6,458,937, which ishereby expressly incorporated by reference in its entirety, describesseveral cell based protocols for synthesizing saccharides andglycoconjugates. By one approach to synthesize the specificityexchangers described herein saccharides and glycoconjugates are firstmade by (a) contacting a cell with a first monosaccharide, and (b)incubating the cell under conditions whereby the cell (i) internalizesthe first monosaccharide, (ii) biochemically processes the firstmonosaccharide into a second saccharide, (iii) conjugates the saccharideto a carrier to form a glycoconjugate, and (iv) extracellularly expressthe glycoconjugate to form an extracellular glycoconjugate comprising aselectively reactive functional group. By then reacting theglycoconjugate containing the functional group with a specificityexchanger comprising a reactive functional group, the glycoconjugate andspecificity exchanger are joined. Subject compositions can includecyto-compatible monosaccharides comprising a nitrogen or ether linkedfunctional group, for example, that are selectively reactive withsimilar groups present on a specificity exchanger.

By another approach, the saccharides and glycoconjugates can besynthesized by a) contacting a cell with a first monosaccharidecomprising a first functional group, and b) incubating the cell underconditions whereby the cell (i) internalizes the first monosaccharide,(ii) biochemically processes the first monosaccharide into a secondmonosaccharide which comprises a second functional group, (iii)conjugates the second monosaccharide to a carrier to form aglycoconjugate comprising a third functional group, and (iv)extracellularly expresses the glycoconjugate to form an extracellularglycoconjugate comprising a fourth, selectively reactive, functionalgroup.

Extracellular glycoconjugates synthesized by the above method may bepresented in multiple forms such as membrane-associated (e.g., amembrane bound glycolipid or glycoprotein), associated withcell-proximate structures (e.g., extracellular matrix components orneighboring cells), or in a surrounding medium or fluid (e.g., as asecreted glycoprotein). The selective reactivity of the fourthfunctional group permits selective targeting of the glycoconjugate aspresented by the cell. For example, fourth functional groups of surfaceassociated glycoconjugates can provide a reactivity that permits theselective targeting of the glycoconjugate in the context of theassociated region of the cell surface. Preferentially reactivity may beaffected by a more reactive context. For example, theglycoconjugate-associated fourth functional group provides greateraccessibility, greater frequency or enhanced reactivity as compared withsuch functional groups present proximate to the site of, but notassociated with the glycoconjugate. In a preferred embodiment, thefourth functional group is unique to the region of glycoconjugatepresentation.

The selective reactivity provided by the fourth functional group maytake a variety of forms including nuclear reactivity, such as theneutron reactivity of a boron atom, and chemical reactivity, includingcovalent and non-covalent binding reactivity. In any event, the fourthfunctional group should be sufficient for the requisite selectivereactivity. A wide variety of chemical reactivities may be exploited toprovide selectivity, depending on the context of presentation. Forexample, fourth functional groups applicable to cell surface-associatedglycoconjugates include covalently reactive groups not normallyaccessible at the cell-surface, including alkenes, alkynes, dienes,thiols, phosphines and ketones. Suitable non-covalently reactive groupsinclude haptens, such as biotin, and antigens such as dinitrophenol.

In more embodiments, the nature of the expressed glycoconjugate is afunction of the first monosaccharide, the cell type and incubationconditions. In these embodiments, the resident biochemical pathways ofthe cell act to biochemically process the first monosaccharide into thesecond monosaccharide, conjugate the second monosaccharide to anintracellular carrier, such as an oligo/polysaccharide, lipid orprotein, and extracellularly express the final glycoconjugate.Alternatively, the expressed glycoconjugate may also be a function offurther manipulation. For example, the fourth functional group mayresult from modifying the third functional group as initially expressedby the cell. For example, the third functional group may comprise alatent, masked or blocked group that requires a post-expressiontreatment, e.g., chemical cleavage or activation, in order to generatethe fourth functional group. Such treatment may be effected by enzymesendogenous to the cell or by exogenous manipulation. Hence, the thirdand fourth functional groups may be the same or different, depending oncellular or extracellular processing events.

As indicated, a functional group can be a masked, latent, inchoate ornascent form of another functional group. Examples of masked orprotected functional groups and their unmasked counterparts are providedin TABLE 3. Masking groups may be liberated in any convenient way; forexample, ketal or enols ether may be converted to corresponding ketonesby low pH facilitated hydrolysis. Alternatively, many specific enzymesare known to cleave specific protecting groups, thereby unmasking afunctional group.

TABLE 3 Masking group Unmasked group dialkyl ketal ketone acetalaldehyde enol ether ketone or aldehyde oxime ketone hydrazone ketonethioester thiol cobalt-complexed alkyne alkyne

In contrast, the nature of the intracellular glycoconjugate (comprisingthe third functional group) is generally solely a function of the firstmonosaccharide, the cell type and incubation conditions. For example,the first and second monosaccharides and the saccharide moietyincorporated into the intracellular glycoconjugate (as well as thefirst, second and third functional groups) may be the same or differentdepending on cellular processing events. For example, the firstmonosaccharide or functional group, cell and conditions may interact toform a chemically distinct second monosaccharide or functional group,respectively. For example, many biochemical pathways are known tointerconvert monosaccharides and/or chemically transform variousfunctional groups. Hence, predetermined interconversions are provided bya first monosaccharide, cell and incubation condition selection.

The first monosaccharide is selected to exploit permissive biochemicalpathways of the cell to effect expression of the extracellularglycoconjugate. For example, many pathways of sialic acid biosynthesisare shown to be permissive to a wide variety of mannose and glucosederivatives. The first functional group may be incorporated into thefirst monosaccharide in a variety of ways. In preferred embodiments, thefunctional group is nitrogen or ether linked.

A wide variety of cells may be used according to the disclosed methodsincluding eukaryotic, especially mammalian cells (e.g., pigs, mice, andNew World monkeys) and prokaryotic cells. The cells may be in culture,e.g., immortalized or primary cultures, or in situ, e.g., resident inthe organism.

The methods herein are also directed to forming products attached to thecell. Generally, these methods involve expressing an extracellularglycoconjugate as described above wherein the expressed glycoconjugateis retained proximate to the cell; for example, by being bound tomembrane or extracellular matrix components. Then the fourth functionalgroup is contacted with an agent which selectively reacts with thefourth functional group to form a product.

A wide variety of agents may be used to generate a wide variety ofproducts. Generally, agent selection is dictated by the nature of thefourth functional group and the desired product. For example, withchemically reactive fourth functional groups, the agent provides a fifthfunctional group that selectively chemically reacts with the fourthfunctional group. For example, where the fourth functional group is aketone, suitable fifth functional groups include hydrazines,hydroxylamines, acyl hydrazides, thiosemicarbazides andbeta-aminothiols. In other embodiments, the fifth functional group is aselective noncovalent binding group, such as an antibody idiotope. Inyet other embodiments, suitable agents include radioactivity such asalpha particles which selectively react with fourth functional groupscomprising radiosensitizers such as boron atoms; oxidizers such asoxygen which react with fourth functional groups comprising a surfacemetal complex, e.g., to produce cytotoxic oxidative species; etc.Alternatively, a functional group on the cell surface might have uniqueproperties that do not require the addition of an external agent (e.g.,a heavy metal which serves as a label for detection by electronmicroscopy). Further examples of products formed by the interaction of acell surface functional group and an agent are given in TABLE 4.

TABLE 4 Functional group Agent Product ketone hydrazide hydrazone dienedienophile Diels-Alder adduct thiol alpha-bromo amide thioether boronneutrons radiation biotin avidin biotin-avidin complex dinitrophenol(DNP) anti-DNP antibodies DNP-antibody complex Fluorescein UV lightgreen light iron complex oxygen peroxy radicals

Frequently, the agent comprises an activator moiety, which provides adesired activity at the cell. A wide variety of activator moieties maybe used, including moieties which alter the physiology of the cell orsurrounding cells, label the cell, sensitize the cell to environmentalstimuli, alter the susceptibility of the cell to pathogens or genetictransfection, etc. Exemplary activator moieties include toxins, drugs,detectable labels, genetic vectors, molecular receptors, and chelators.

A wide variety of compositions useful in the disclosed methods areprovided herein. These compositions include cyto-compatiblemonosaccharides comprising a functional group, preferably a nitrogen orether linked functional group, which group is selectively reactive at acell surface. Exemplary functional groups of such compounds includealkynes, dienes, thiols, phosphines, boron and, especially, ketones. Theterm substituted or unsubstituted alkyl is intended to encompass alkoxy,cycloalkyl, heteroalkyl, and similar compounds. Similarly, the termsubstituted or unsubstituted aryl is intended to encompass aryloxy,arylalkyl (including arylalkoxy, etc.), heteroaryl, arylalkynyl, andsimilar compounds. The term substituted or unsubstituted alkenyl isintended to analogously encompass cycloalkenyl, heteroalkenyl, etc.Analogous derivatives are made with other monosaccharides havingpermissive pathways of bioincorporation. Such monosaccharides arereadily identified in convenient cell and protein-based screens, such asdescribed below. For example, functionalized monosaccharidesincorporated into cell surface glycoconjugates can be detected usingfluorescent labels bearing a complementary reactive functional group. Acell-based assay suitable for mechanized high-throughput opticalreadings involves detecting ketone-bearing monosaccharides on cellsurfaces by reaction with biotin hydrazide, followed by incubation withFITC-labeled avidin and then quantitating the presence of thefluorescent marker on the cell surface by automated flow cytometry. Aconvenient protein-based screen involves isolating the glycoconjugate(e.g., gel blots), affinity immobilization, and detecting with thecomplementary reactive probe (e.g., detone-bearing glycoconjugatesdetected with biotin hydrazide), followed by incubation withavidin-alkaline phosphatase or avidin-horseradish peroxidase.Alternatively, monosaccharides bearing unusual functional groups canalso be detected by hydrolysis of the glycoconjugate followed byautomated HPLC analysis of the monosaccharides. The following sectiondescribes several approaches to manufacture the specificity exchangersdescribed herein that utilize methods of chemical synthesis.

Chemical Synthesis

In addition to using enzyme catalyzed methods and cell-based methods,the specificity exchangers comprising saccharides and glycoproteins canbe made using methods directed to chemical synthesis. Examples ofmethods used to synthesize saccharides and glycoconjugates can be foundin Pamela Sears et al., Toward Automated Synthesis of Oligosaccharidesand Glycoproteins, Carbohydrates and Glycobiology 291 Science 2344 (Mar.23, 2001), which is hereby incorporated by reference in its entirety.Most methods of chemical synthesis involve the activation of theanomeric leaving group with a Lewis acid. The Koenigs-Knorr method ofcoupling glycosyl halides, one of the first techniques to gainwidespread usage, is still in common use, and most other glycosidationreagents used to date proceed by the same basic mechanism.

Chemical synthesis of saccharides and glycoconjugates can also beperformed automatically. Generally for automated synthesis, it isconvenient for the reactions to be performed on solid phase. Thisapproach allows the rapid removal of reactants, relatively easypurifications, and (in the case of library construction) the encoding ofthe product either by position (as in a two-dimensional array “chip”format) or, for “mix and split” type library construction, by anaccessory encoding reaction, in which the labels are added to the solidsupport as the chain is extended or by radio frequency-encodedcombinatorial chemistry technology. Hydrophilic supports, such aspolyethylene glycol-based resins, have been used with good success, ashave “hybrid” resins, such as Tentagel, that have a polystyrene corecoated in polyethylene. To a lesser extent, soluble supports, such aspolyethylene glycols and derivatives, have been used in saccharidesynthesis.

Another approach that can be used for saccharide and glycoconjugatesynthesis is a one-pot reaction. One-pot reactions rely on thereactivity profile of different protected sugars to determine thesynthesized product. The reactivity of a sugar is highly dependent onthe protecting groups and the anomeric activating group used. By addingsubstrates in sequence from the most reactive to least reactive, one canassure the predominance of a desired target compound. The key to thisapproach is to have extensive quantitative data regarding the relativereactivities of different protected sugars, which is currently beinggenerated by those with skill in the art of glycomics. These reactionsare typically performed in solution, but in order to facilitate removalof reactants at the end, the final acceptor may be attached to a solidphase.

This approach can be made even more efficient through automation, suchas a computer program. Compared with stepwise solid-phase synthesis, theone-pot approach uses protecting-group manipulation only at the stage ofbuilding block synthesis and thus holds greater potential for automationand for greater diversity of oligosaccharide structures.

Additionally, several other methodologies can be employed to synthesizethe glycopeptides and glycoproteins that are joined to or incorporatedin the specificity exchangers described herein. Several of these methodsare discussed in Pamela Sears et al., Toward Automated Synthesis ofOligosaccharides and Glycoproteins, Carbohydrates and Glycobiology 291Science 2344 (Mar. 23, 2001), which is hereby incorporated by referencein its entirety. By one approach, for example, attachment of saccharidechains to the specificity exchangers described herein is accomplished ina stepwise fashion, beginning from the nonreducing end and proceeding tothe reducing end. As is the case with glycal-based synthetic schemes andthe one-pot strategy outlined above, the ultimate acceptor can be anamino acid, peptide or glycopeptide. For coupling to hydroxylated aminoacids, such as serine or threonine, the chemistry is very much the sameas that used to construct the glycosidic bonds: the activated anomericposition is directly attacked by a deprotected hydroxyl group on thepeptide. In the case of NH₂-linked glycosides, the reducing-end sugar istypically prepared first as a sugar azide, which is then reduced andcoupled to a free aspartate via carbodiimide activation. The acceptorcan be an amino acid, for which the product can be incorporated intosolid-phase peptide synthesis (SPPS) schemes to produce the targetglycopeptide, or it may itself be the final polypeptide. Glycosylatedamino acids bearing typically one to three sugars have been usedsuccessfully in solid phase synthesis of many glycopeptides.

In certain embodiments the glycopeptide containing specificityexchangers described herein can be synthesized by glycosylating thepeptide in a stepwise fashion from the reducing to the nonreducing endthrough chemical or enzymatic methods. Typically, a single glycosylatedpeptide is made by SSPS, the sugar is selectively deprotected, and theoligosaccharide is built up in a stepwise fashion. The singlyglycosylated peptide can be constructed via SPPS, and the sugar can becompletely deprotected to provide the substrate for the action of threesuccessive glycosyltransferases. The synthesis of these glycopeptidescan also be automated.

Extension of glycosylated peptides into glycoproteins can also beaccomplished by a number of approaches. Workers (Allen, P. Z., andGoldstein, I. J., Biochemistry, 1967, 6, 3029; Rude, E., and Delius, M.M., Carbohydr. Res., 1968, 8, 219; Himmelspach, K., et al., Eur. J.Immunol., 1971, 1, 106; Fielder, R. J., et al., J. Immunol., 1970, 105,265) developed several techniques for conjugation of carbohydrates toprotein carriers, for example. Most of them suffered by introducing anantigenic determinant in the linker itself, resulting in generation ofpolyclonal antibodies. Kabat (Arakatsu, Y., et al., J. Immunol., 1966,97, 858), and Gray (Gray, G. R., Arch. Biochem. Biophys. 1974, 163, 426)developed conjugation methods that relied on oxidative or reductivecoupling, respectively, of free reducing oligosaccharides. The maindisadvantage of these techniques, however, is that the integrity of thereducing end of the oligosaccharide was compromised. In 1975 Lemieuxdescribed the use an 8-carbomethoxy-1-octanol linker (Lemieux, R. U., etal., J. Am. Chem. Soc., 1975, 97, 4076) which alleviated the problem oflinker antigenicity and left the entire oligosaccharide intact. Equallyeffective in producing glycoconjugates was the allyl glycoside methoddescribed by Bernstein and Hall. (Bernstein, M. A., and Hall, L. D.,Carbohydr. Res., 1980, 78, C1.) In this technique the allyl glycoside ofthe deblocked sugar is ozonized followed by a reductive workup. Theresultant aldehyde is then reductively coupled to a protein carrier withsodium cyanoborohydride.

Short peptides can also be coupled to larger ones by “native peptideligation” strategies. Easier approaches to glycoprotein synthesis can beachieved through cell based methods, however. The glycans produced bythis method will be determined by many factors, including the localprotein structure around the glycosylation site and the relative amountsof glyco-processing enzymes produced in the cell. Many of these factorsalso vary with the cell line, so a glycoprotein produced in one cellline may have different glycosylation than the same protein produced inanother cell line.

The resulting products however can be used as a starting point for manyschemes in which the sugar chain is digested down to a simplehomogeneous core and then reelaborated enzymatically. For example,N-glycosylated proteins can have the glycans digested down to theinnermost N-acetylglucosamine by using endoglycosidases, thus convertinga heterogeneous population to a homogeneous one in which eachglycosylation site has only a single sugar attached. These simpleglycoproteins can then be elaborated enzymatically to increase the sizeand complexity of the glycan by using glycosyltransferases orendoglycosidase-catalyzed transglycosylation. The transglycosidaseapproach is limited by the substrate specificity of theendoglycosidases, which are enzymes that cleave between the innermostN-acetylglucosamine residues of N₂-linked oligosaccharides. In certainembodiments the endoglycosidase can be endoglycosidase M from Mucorhiemalis, which accepts a wide range of high-mannose-, hybrid-andcomplex-type glycans.

Another option is to remove the glycosylated sections by using proteasesand then reattach short, chemically synthesized glycopeptides in theirplace. This ligation can be accomplished enzymatically through the useof proteases or inteins, self-splicing polypeptides that are able toexcise themselves from proteins posttranslationally. In the latter case,the peptide segment to be replaced is substituted at the genetic levelwith the sequence encoding the intein.

Proteases can catalyze peptide synthesis using either the thermodynamicapproach or the kinetic approach. In the thermodynamic approach,peptides are condensed to form the larger product typically byprecipitation of the product or by conducting the reaction in a solventwith low water activity. A more useful approach, as far as enzymeactivity, stability, and solubility are concerned, is the kineticapproach, in which a peptide ester undergoes a competition betweenhydrolysis and aminolysis. The ratio of aminolysis to hydrolysis can beimproved by adding an organic cosolvent to lower the water concentrationand suppress amine ionization, by increasing the amine nucleophileconcentration, or by modifying the enzyme active site. With regard toenzyme modification, the conversion of the active-site serine of serineproteases to a cysteine has been shown to be highly effective forcreating a peptide ligase. Glycosylation of proteins has long been knownto render them less susceptible to protease activity, and so it might beinferred that glycopeptides would be difficult to couple usingproteases. A systematic study of subtilisin-catalyzed synthesis ofglycopeptides, however, reveals that the protease could coupleglycopeptides successfully, provided that the glycosylation site was notat the forming bond and that the coupling yields improved as theglycosylation site was placed further away from it. One of the mosteffective and practical glycopeptide ester leaving groups is thebenzyl-type ester generated from a modified Rink amide resin and cleavedwith trifluoroacetic acid.

An alternate approach is to use intein-mediated coupling ofglycopeptides to larger proteins. It is possible to intervene in thenatural splicing reaction by removing the COOH-terminal extein, thenallowing the reaction to be completed with an exogenously addednucleophile, which may be a glycopeptide. As in the native peptideligation strategy, the peptide preferably contains a cysteine at theNH₂-terminus.

Glycoprotein purification procedures can be very similar to thepurification of unglycosylated proteins. The first step in glycoproteinpurification is usually to solublize the glycoprote in. Glycoproteinsthat are secreted into the media are relatively easy to purify if serumfree media has been used to grow the cells. Glycoproteins that remaintrapped in a vesicle (as seen with chicken Thy-1) can be solublized withdetergents. Once in detergent, the proteins can be dialyzed againstaqueous buffers.

After solublizing the glycoprotein, various chromatographic purificationschemes can be used to purify it. In certain embodiments, LectinAffinity Chromatography can be used. Lectins are non-immune proteins orglycoproteins that bind to specific saccharides and glycoconjugates withhigh affinity. Because of their binding specificity, lectins show arange of specificities for carbohydrates and glycoconjugates. Theselectins can easily be immobilized onto a variety of supports and usedfor affinity chromatography. Once coupled, lectins are stable with mostof the buffers.

Research carried out by Arya, et al., has lead to development of anautomated, multi-step, solid-phase strategy for the parallel synthesisof artificial glycopeptide libraries. (Arya, P. et. al., 7 Med. Chem.Lett. 1537, 1997, herein expressly incorporated by reference in itsentirety). In some embodiments, the specificity exchangers describedherein are constructed using this strategy. FIG. 1 illustrates thisapproach.

Accordingly, different α- or β-carbon linked carbohydrate based aldehydeand carboxylic acid derivatives, protected as acetates (see 18.1 inFIG. 1) can be incorporated either at the N-terminal moiety or at theinternal amide nitrogen of short peptides/pseudopeptides (e.g.,specificity domains or specificity domains joined to an antigenicdomain) in a highly flexible and controlled manner. The chain length ofthe C-glycoside can be varied and the carbohydrate moiety can besynthesized in either the pyranose or furanose form. Monosaccharides andtheir derivatives are not the only available carbohydrate buildingblocks. In certain embodiments, disaccharides and higher orderoligosacccharides can also be used as carbohydrate building blocks.C-Glycosides are generally more stable to enzymatic and acid/basehydrolysis than their oxygen counterparts. This method is more versatilethan the glycosylated amino acid building block in which the choice ofamino acids is limited.

Using this approach, libraries of artificial glycopeptides can bereadily synthesized for probing carbohydrate-protein interactions.Several “working models” that display multiple copies of carbohydrateshave been developed (see 18.2, 18.3, and 18.4 in FIG. 1) while thedipeptide scaffold may contribute to secondary interactions with thebiological target. (Arya, P. et al., 8 Med. Chem. Lett. 1127, 1998;Arya, P. et al., 7 Med. Chem. 2823, 1999).

Initially, artificial glycopeptides were synthesized by a convergentstrategy on a peptide synthesizer. (Kutterer, et. al., 1 J. Comb. Chem.28, 1999). The synthesis of these artificial glycopeptide libraries hasbeen successfully transferred to a fully automated multiple organicsynthesizer and each step in the synthesis was optimized. (Arya, P. etal., 2 Comb. Chem. 120, 2000). This methodology involves coupling anamino acid to a solid-support resin such as Rink amide MBHA resin orTentaGel derivatized Rink amide resin. After removal of the protectinggroup on the amino acid, the sugar aldehyde undergoes reductiveamination (see 18.3 and 18.4 in FIG. 1) with the resin bound amino groupfollowed by amino acid coupling of the second amino acid. Afterdeprotection of the amino acid, a second reductive amination can occurand/or a sugar acid can be coupled. The sugar moieties are thendeacetylated, and the compounds are cleaved from the resin. Thesynthesis of a 96 compound library can be obtained from just 24dipeptides and two sugar aldehydes. (See Randell, Karla D., et al.,High-throughput Chemistry toward Complex Carbohydrates andCarbohydrate-like Compounds, National Research Council of Canada,publication no. 43876, Feb. 13, 2001).

A recent article describes another approach that can be used tomanufacture the specificity exchangers described herein. The synthesisof multivalent cyclic neoglycopeptides has been accomplished. (SeeWittmann, V.; Seeberger, et al., 39 Chem. Int. Ed. 4348, 2000, hereinexpressly incorporated by reference in its entirety). A newurethane-type linker based on the Alloc protecting group was developedfor the glycosylation reaction, which proceeds virtually quantitatively.A library of cyclic peptides (e.g., specificity exchangers) can besynthesized using the split and mix method on TentaGel resin linked viathe Sieber linker. FIG. 2 illustrates this approach.

The synthesis reaction shown in FIG. 2 can be monitored by withdrawal ofa small amount of resin from the well and analysis by HPLC incombination with electrospray mass spectrometry. The p-nitrophenylcarbonate derivative of the sugar moiety (see 19.2 in FIG. 2) wasattached to three points of the cyclic peptide in one step using afive-fold excess (per attachment point) of the sugar in the presence ofDIPEA. A library of eighteen cyclic neoglycopeptides (see 19.3 in FIG.2) can be efficiently synthesized. This methodology can be applied tothe synthesis of many different libraries by varying the distancesbetween the carbohydrate moieties as well as the carbohydrate moietyitself. The section below discusses the incorporation of linkers to thespecificity exchangers and/or saccharides or glycoconjugates.

Linkers

In certain embodiments, the saccharide or glycoconjugates can be joinedto the specificity exchangers through linkers or by association with acommon carrier molecule, as discussed previously. In some embodimentslinkers are used to join saccharides to at least one amino acid of thespecificity exchangers. In general the term “linkers” refers to elementsthat promote flexibility of the molecule, reduce steric hindrance, orallow the specificity exchanger to be attached to a support or othermolecule. Any suitable linker can be used to attach the saccharide andor glycoconjugate to a specificity exchanger. In certain embodiments thelinker can be polyethylene glycol.

Other types of linkers that can be incorporated with a specificityexchanger include avidin or streptavidin (or their ligand—biotin).Through a biotin-avidin/streptavidin linkage, multiple specificityexchangers can be joined together (e.g., through a support, such as aresin, or directly) or individual specificity domains can be joined to asaccharide or glycoconjugate.

Another example of a linker that can be included in a specificityexchanger is referred to as a “λ linker” because it has a sequence thatis found on λ phage. Preferred λ sequences are those that correspond tothe flexible arms of the phage. These sequences can be included in aspecificity exchanger (e.g., between the specificity domain and thesaccharide or glycoconjugate or between multimers of the specificityand/or saccharides and glycoconjugates) so as to provide greaterflexibility and reduce steric hindrance. The Example below describes themanufacture of a specificity exchanger comprising a plurality ofsaccharides.

EXAMPLE 1

Two different ligand/receptor specificity exchangers having identicalspecificity domains (approximately 20 amino acids long) comprising afragment of the fibrinogen gamma-chain are produced using standardtechniques in peptide and glycoconjugate synthesis. The firstspecificity exchanger (Specificity Exchanger 1) includes an antigenicdomain having a peptide obtained from the poliovirus. This polioviruspeptide is recognized by antibodies present in human sera obtained froman individual that had been inoculated against polio. The secondspecificity exchanger (Specificity Exchanger 2) is identical toSpecificity Exchanger 1, except that a saccharide antigen, (e.g.,gal-α-1-3 gal) has been added to the specificity exchanger using one ofthe techniques described above or an another commonly used approach.This gal antigen is also recognized by antibodies present in the humansera obtained from the individual having anti-polio peptide antibodies,described above. Because Specificity Exchangers 1 and 2 have identicalspecificity domains, which would be expected to bind immobilized ClfAreceptor equally well, the ability of the saccharide-containingantigenic domain to recruit more antibodies from human sera than theantigenic domain lacking the gal antigen can be directly analyzed in asandwich-type plate assay.

Accordingly, serial dilutions of the two ligand/receptor specificityexchangers above are prepared and recombinant ClfA is passively adsorbedat 10 μg/ml to 96 well microtiter plates in 50 mM sodium carbonatebuffer, pH 9.6, overnight at 4° C. The diluted ligand/receptorspecificity exchangers are then applied to the ClfA-bound plates for 60minutes at 4° C. with gentle rocking. In some wells, a blocking agentsuch as BSA is added prior to addition of the specificity exchanger todecrease the non-specific binding. Next, the plates are washed fourtimes with 2 ml of 50 mM sodium carbonate buffer, pH 9.6, to remove anyunbound specificity exchanger. After washing, 1 ml of human seraobtained from a subject that has antibodies to both the polioviruspeptide and the gal antigen is added to the wells in 1 ml of 50 mMsodium carbonate buffer, pH 9.6 (i.e., 1:1 ratio), and the plates arerocked gently overnight at 4° C. Again BSA may be included to blocknon-specific binding. The washing steps performed previously arerepeated so as to remove any non-specifically bound antibody.

Several methods of analysis can then be employed. By one approach, thebound antibody is eluted from the specificity exchanger using a typicalantibody elution buffer (e.g., Glycine C1 pH 2.5; see Yarmush et al.,Biotechnol. Prog. 8:168-178 (1992), hereby expressly incorporated byreference in its entirety) and the absorbance of the eluant is detectedspectrophotometrically. In some cases a dye is employed to improve thelevel of detection. Alternatively, the eluant can be blotted to amembrane (e.g., using a dot blot manifold) and the amount of protein inthe eluant can be quantified using silver staining, fluorescence, or adye-based assay. The eluant can also be applied to a polyacrylamide gel,separated by electrophoresis, and stained or transferred to a membrane,which is then subjected to Western blot using peroxidase labeledantibodies specific for the human immunoglobulins G, A, and M.(peroxidase labeled polyvalent human immunoglobulins available fromSigma). Additionally, the amount of antibody from human sera that boundthe specificity exchangers can also be determined in situ (i.e., withouteluting from the plate), using typical sandwich-type assays that employthe peroxidase labeled polyvalent antibodies, described above. By thesemethods of analysis, the plates are developed by incubation withdinitro-phenylene-diamine (Sigma) and the absorbance is analyzed.

It is expected that the analysis described above will confirm thatsignificantly more antibodies from human sera will bind to the antigenicdomain composed of the polio peptide and the gal antigen (i.e.,Specificity Exchanger 2) than the polio peptide alone (i.e., SpecificityExchanger 1). Because the ClfA receptor is present on pathogens such asStaphylococcus, this assay will also confirm that specificity exchangersthat comprise the gal antigen (e.g., gal-α-1-3 gal) are more effectiveat recruiting the natural antibodies present in a subject and,therefore, are more effective at redirecting these antibodies to apathogen. The next Example describes an approach that was used tosynthesize several glycosylated ligand/receptor specificity exchangers.

EXAMPLE 2

Several glycosylated ligand/receptor specificity exchangers (seeTABLE 1) comprising a specificity domain corresponding to a CD4 receptorregion that interacts with the HIV-1 glycoprotein 120 (see Mizukami T.,Fuerst T. R., Berger E. A. & Moss B., Proc Natl Acad Sci USA. 85, 9273-7(1988), herein expressly incorporated by reference in its entirety, weresynthesized on solid phase. The peptides were produced in an automaticsynthesis robot using Fmoc chemistry (see Ed. Chan W. C. & White P. D.Fmoc solid phase peptide synthesis-α practical approach (2000) Oxforduniversity press., herein expressly incorporated by reference in itsentirety). Each peptide, still attached to the solid support (resin),was divided in a minor and a major fraction. The minor peptide fractionswere cleaved off the resin by treatment with TFA, while the majorfractions were left attached to the resin, awaiting glycosylation. Thecleaved peptides were analysed by reversed phase HPLC (λ=220 nm) tocheck the purity. After analysis, the cleaved peptides were lyophilised.

A reagent including the sugar Gal-α1-3Gal has been shown to absorb humananti-Gal-α 1-3Gal antibodies (Rieben R., von Allmen E., Korchagina E.Yu. Et al. Xenotransplantation. 2, 98-106 (1995), herein expresslyincorporated by reference in its entirety). The formula forGal-α1-3Gal-β is provided below as Formula 1.

The sugar reagent (Galα1-3Galβ1-O(CH₂)₃NH₂ (Lectinity corp., product no.88)) was coupled to the side chain of an aspartic acid (Formula 2):

The amino acid was protected both at the N-terminal and C-terminal ends.A covalent bond between the sugar amino group and the amino acidcarboxyl group, was formed. (See Synthesis 1).

The coupling reaction was monitored by analysing samples from thereaction mixture by reversed phase HPLC using λ=266 nm since the Fmocprotecting group absorbs UV light strongly at this wavelength. Thecoupling reaction was stopped after ˜4-6 h. The glyco-amino acid waspurified by reversed phase HPLC (λ=266 nm). The purified glyco-aminoacid was lyophilised.

Next, deprotection of the glyco-amino acid was performed. To allowcoupling of the glyco-amino acid to the CD4 peptides, theOtBu-protecting group was cleaved off the glyco-amino acid C-terminus,by treatment with TFA (see Synthesis 2).

The deprotection was monitored by reversed phase HPLC (λ=266 nm). After˜2 h, the deprotection was stopped. Most of the TFA was evaporated bynitrogen gas. The remaining solution was diluted 1/10 with water. Thedeprotected glyco-amino acid was purified by reversed phase HPLC (λ=266nm). The purified and deprotected glyco-amino acid was lyophilised.

Following deprotection of the glyco-amino acid, coupling to the CD4 andCDR specificity domain peptides was performed. The glyco-amino acid wascovalently linked to the N-terminal ends of the resin-bound specificitydomains using Fmoc chemistry (see Synthesis 3). The coupling reactionwas stopped after ˜6 h.

The glycosylated specificity exchangers were deprotected and cleaved offtheir solid supports by treatment with TFA. The cleaved glycosylatedspecificity exchangers were analysed by reversed phase HPLC (λ=220 nm)to check the purity in comparison with the correspondingnon-glycosylated peptides. Glycosylation-specific peaks were purified byreversed phase HPLC (λ=220 nm). The purified glycosylated specificityexchangers were lyophilised. After lyophilization, a fraction of eachglycosylated specificity exchangere was analysed by MALDI-MS to verifyits identity. The following Example describes several cellular-basedcharacterization assays that can be performed to determine whether aligand/receptor specificity exchanger inhibits the proliferation of apathogen.

EXAMPLE 3

One type of pathogen-based characterization assay involves the bindingof ligand/receptor specificity exchangers to bacteria disposed on asupport. As in Example 1, separate assays are performed to compare thebinding affinities of Specificity Exchangers 1 and 2. Bacteria thatproduce ClfA (e.g., Staphylococcus aureus, or Escherichia coli.) aregrown in culture or on several agar plates in a suitable growth media(e.g., LB broth, blood broth, LB agar or blood agar). The cells aregrown to confluency so as to produce a solid bacterial lawn. Next,several dilutions of Specificity Exchanger 1 and Specificity Exchanger 2are added to separate plates. For example, different plates receive 500mg, 1 mg, 5 mg, 10 mg, 25 mg, and 50 mg of either Specificity Exchanger1 or 2 in a total volume of 200 μl of PBS. The plates are incubated at37° C. for at least 4 hours.

Subsequently, the non-bound ligand/receptor specificity exchangers areremoved with successive washes with PBS (e.g., 3 washes with 2 ml of PBSper wash). Next, serial dilutions of the human sera used in Example 1(i.e., it contains antibodies to both the polio peptide and the galantigen) are added to the plates (e.g., 1:100-1:1000 dilutions of humansera are provided). After a 60 minute incubation, the plates are washedwith PBS (e.g., 3 washes with 2 ml of PBS per wash) to remove unboundprimary antibody. The bacterial proteins, specificity exchangers, andhuman antibodies are then transferred to a membrane. Appropriatecontrols include the membrane itself, bacterial proteins transferred tothe membrane without a ligand/receptor specificity exchanger butcontaining antibodies from human sera, and bacterial proteinstransferred to the membrane with ligand/receptor specificity exchangerbut no antibodies from human sera.

The amount of antibodies redirected to the bacteria can then beascertained by using the peroxidase labeled antibodies specific for thehuman immunoglobulins G, A, and M, as described in Example 1. Anappropriate dilution of the secondary antibody is contacted with themembrane for 60 minutes and the non-bound secondary antibody is washedfrom the membrane with PBS (e.g., 3 washes with 2 ml of PBS per wash).The bound secondary antibody is then detected by incubating the membranewith dinitro-phenylene-diamine (Sigma).

The data will show that the specificity exchanger comprising the galantigen (Specificity Exchanger 2) redirected antibodies present in humansera to the bacterial pathogen more efficiently than SpecificityExchanger 1. This example also demonstrates that specificity exchangersthat comprise a plurality of saccharides or glycoconjugates (e.g.,gal-α-1-3 gal) will be more effective at redirecting antibodies presentin a subject to a pathogen in vivo. The next Example describes apathogen-based characterization assay that was performed to evaluate theability of glycosylated ligand/receptor specificity exchangers tointeract with HIV.

EXAMPLE 4

Glycosylated ligand/receptor specificity exchangers specific for HIVwere produced according to the approaches described in Example 2. Toevaluate the ability of glycosylated specificity exchangers to bindhuman Gal-alpha1,3-Gal-specific antibodies, glycosylated andnon-glycosylated versions of the same ligand/receptor specificityexchanger were coated on solid phase of microtitre plates. Four humansera were allowed to bind to the coated peptides, and an enzyme labeledanti-human antibody indicated bound human antibodies. The results showedthat only the glycosylated peptides were able to bind human antibodies(human sera IB72; FIG. 3).

To further evaluate the ability of the glycosylated specificityexchangers to bind to a pathogen, a glycosylated peptide competitiveassay was performed using the most reactive human sera (IB72). In brief,Gal-alpha1,3-Gal-labeled bovine serum albumin (Gal-BSA) was coated onto96-well microplates in sodium carbonate buffer (pH: 9.6) at +4° C.overnight. Dilutions of human sera and dilutions of glyco-peptides(glycosylated HIV-specific ligand/receptor specificity exchangers orGal-BSA) or non-glycosylated peptides (HIV-specific ligand/receptorspecificity exchangers or BSA) were preincubated in phosphate-bufferedsaline (PBS) containing 1% bovine albumin, 2% goat serum and 0.05% Tween20 at 37° C. for 1 h. The mixture was then added to the coated platesand incubated at 37° C. for 1 h, then washed 3 times withphosphate-buffered saline (PBS) containing 0.05% Tween 20 (pH: 7.4).

Bound antibodies were indicated with goat anti-human polyvalent Ig,conjugated with alkaline phosphatase. The plates were incubated andwashed as described above. Plates were developed with phosphatasesubstrate at room temperature for 30 min, stopped with 1 M NaOH. Opticaldensity (OD) at 405 nm/650 nm was determined to quantify the inhibition.The results are provided in FIG. 4, which shows that the human antibodybinding to Gal-BSA could only be inhibited by either Gal-BSA or theglycosylated peptide. Thus, specificity exchangers that bindGal-alpha1,3-Gal-specific antibodies had been generated. Gal-BSA mixedwith human sera in the same conditions as mentioned above was used aspositive control and 100% inhibition was observed.

The following example will demonstrate that specificity exchangerscomprising a plurality of saccharides or glycoconjugates (e.g.,gal-α-1-3 gal) are more effective at redirecting antibodies present in asubject to a pathogen in vivo.

EXAMPLE 5

There are many animal models that are suitable for evaluating theability of a ligand/receptor specificity exchanger to inhibit pathogenicinfection. Mice are preferred because they are easy to maintain and aresusceptible to bacterial infection, viral infection, and cancer.Chimpanzees are also preferred because of their close geneticrelationship to humans.

To test the ability of a ligand/receptor specificity exchanger to treata bacterial infection in mice, the following characterization assay canbe performed. Several female CF-1 outbred mice (Charles RiversLaboratories) of approximately 8 weeks of age and 25 gram body mass areinoculated intraperitoneally with overnight cultures of Staphylococcusaureus. Blood samples are drawn from the mice and tests are conducted toverify that Staphylococcus aureus is present in the subjects.

The infected mice are injected with a suitable amount of eitherSpecificity Exchanger 1 or 2, as described in Examples 1 and 2. A smallsample (e.g., 0.5 mL) of the human serum used in Examples 1 and 2 isalso injected into the infected mice. For various time points after theinjection of the human serum for up to two weeks, the mice are monitoredfor the presence and prevalence of Staphylococcus aureus. The progressor decline in Staphylococcus aureus infection is plotted.

The data will show that Specificity Exchanger 2 more efficientlyinhibited the proliferation of Staphylococcus aureus than SpecificityExchanger 1, verifying that the presence of the gal antigen was moreefficient at redirecting the human antibodies present in the subject tothe pathogen. The section below describes several pharmaceuticalscomprising specificity exchangers that comprise saccharides and/orglycoconjugates.

Pharmaceuticals Comprising Specificity Exchangers that CompriseSaccharides and/or Glycoconjugates

The specificity exchangers described herein are suitable forincorporation into pharmaceuticals for administration to subjects inneed of a compound that treats or prevents infection by a pathogen.These pharmacologically active compounds can be processed in accordancewith conventional methods of galenic pharmacy to produce medicinalagents for administration to mammals including humans. The activeingredients can be incorporated into a pharmaceutical product with andwithout modification. Further, the manufacture of pharmaceuticals ortherapeutic agents that deliver the pharmacologically active compoundsof this invention by several routes are aspects of the presentinvention. For example, and not by way of limitation, DNA, RNA, andviral vectors having sequences encoding a specificity exchangerdescribed herein are used with embodiments of the invention. Nucleicacids encoding the embodied specificity exchangers can be administeredalone or in combination with other active ingredients.

The specificity exchangers can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with thepharmacologically active ingredients described herein. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohols, gum arabic, vegetable oils, benzylalcohols, polyetylene glycols, gelatine, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Many more vehicles that can be used are described inRemmington's Pharmaceutical Sciences, 15th Edition, Easton:MackPublishing Company, pages 1405-1412 and 1461-1487(1975) and The NationalFormulary XIV, 14th Edition, Washington, American PharmaceuticalAssociation (1975), herein incorporated by reference. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like so long as theauxiliary agents does not deleteriously react with the specificityexchangers.

The effective dose and method of administration of a particularpharmaceutical having a specificity exchanger that comprises a pluralityof saccharides and/or glycoconjugates can vary based on the individualneeds of the patient and the treatment or preventative measure sought.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation). For example, the effective dose of a specificity exchangercan be evaluated using the characterization assays described above. Thedata obtained from these assays is then used in formulating a range ofdosage for use with other organisms, including humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with no toxicity. The dosage varieswithin this range depending upon type of specificity exchanger, thedosage form employed, sensitivity of the organism, and the route ofadministration.

Normal dosage amounts of a specificity exchanger can vary fromapproximately 1 to 100,000 micrograms, up to a total dose of about 10grams, depending upon the route of administration. Desirable dosagesinclude about 250 mg-1 mg, about 50 mg-200 mg, and about 250 mg-500 mg.

In some embodiments, the dose of a specificity exchanger preferablyproduces a tissue or blood concentration or both from approximately 0.1μM to 500 μM. Desirable doses produce a tissue or blood concentration orboth of about 1 to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about 10 μM to about 500 μM. Althoughdoses that produce a tissue concentration of greater than 800 μM are notpreferred, they can be used. A constant infusion of a specificityexchanger can also be provided so as to maintain a stable concentrationin the tissues as measured by blood levels.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that can be taken into account include theseverity of the disease, age of the organism being treated, and weightor size of the organism, diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Short acting pharmaceutical compositions are administered dailyor more frequently whereas long acting pharmaceutical compositions areadministered every 2 or more days, once a week, or once every two weeksor even less frequently.

Routes of administration of the pharmaceuticals include, but are notlimited to, topical, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Transdermal administration isaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the specificity exchangers to penetrate the skin. Parenteralroutes of administration include, but are not limited to, electrical ordirect injection such as direct injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

Compositions having the specificity exchangers described herein that aresuitable for transdermal or topical administration include, but are notlimited to, pharmaceutically acceptable suspensions, oils, creams, andointments applied directly to the skin or incorporated into a protectivecarrier such as a transdermal device (“transdermal patch”). Examples ofsuitable creams, ointments, etc. can be found, for instance, in thePhysician's Desk Reference. Examples of suitable transdermal devices aredescribed, for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989to Chinen, et al., herein expressly incorporated by reference in itsentirety.

Treatment and Prevention of Disease Using a Specificity Exchanger thatComprises a Plurality of Saccharides and/or a Glycoconjugate

Pharmaceuticals comprising the specificity exchangers described hereincan be administered to a subject in need to treat and/or preventinfection by a pathogen that has a receptor. Such subjects in need caninclude individuals at risk of contacting a pathogen or individuals whoare already infected by a pathogen. These individuals can be identifiedby standard clinical or diagnostic techniques.

By one approach, for example, a subject suffering from a bacterialinfection is identified as a subject in need of an agent that inhibitsproliferation of a pathogen. This subject is then provided atherapeutically effective amount of a specificity exchanger describedherein. The specificity exchanger used in this method comprises aspecificity domain that interacts with a receptor present on thebacteria (e.g., extracellular fibrinogen binding protein (Efb), collagenbinding protein, vitronectin binding protein, laminin binding protein,plasminogen binding protein, thrombospondin binding protein, clumpingfactor A (ClfA), clumping factor B (ClfB), fibronectin binding protein,coagulase, and extracellular adherence protein). The specificityexchanger also comprises an antigenic domain that has a plurality ofsaccharides and/or glycoconjugates, which are recognized by high titerantibodies present in the subject in need. It may also be desired toscreen the subject in need for the presence of high titer antibodiesthat recognize the antigenic domain prior to providing the subject thespecificity exchanger. This screening can be accomplished by EIA orELISA using immobilized antigenic domain or specificity exchanger, asdescribed above.

Similarly, a subject in need of an agent that inhibits viral infectioncan be administered a specificity exchanger that recognizes a receptorpresent on the particular etiologic agent. Accordingly, a subject inneed of an agent that inhibits viral infection is identified by standardclinical or diagnostic procedures. Next, the subject in need is provideda therapeutically effective amount of a specificity exchanger thatinteracts with a receptor present on the type of virus infecting theindividual. As above, it may be desired to determine whether the subjecthas a sufficient titer of antibody to interact with the antigenic domainof the specificity exchanger prior to administering the specificityexchanger.

In the same vein, a subject in need of an agent that inhibits theproliferation of cancer can be administered a specificity exchanger thatinteracts with a receptor present on the cancer cell. For example, asubject in need of an agent that inhibits proliferation of cancer isidentified by standard clinical or diagnostic procedures; then thesubject in need is provided a therapeutically effective amount of aspecificity exchanger that interacts with a receptor present on thecancer cells infecting the subject. As noted above, it may be desired todetermine whether the subject has a sufficient titer of antibody tointeract with the antigenic domain of the specificity exchanger prior toadministering the specificity exchanger.

The specificity exchangers described herein can also be administered tosubjects as a prophylactic to prevent the onset of disease. Virtuallyanyone can be administered a specificity exchanger described herein forprophylactic purposes, (e.g., to prevent a bacterial infection, viralinfection, or cancer). It is desired, however, that subjects at a highrisk of contracting a particular disease are identified and provided aspecificity exchanger. Subjects at high risk of contracting a diseaseinclude individuals with a family history of disease, the elderly or theyoung, or individuals that come in frequent contact with a pathogen(e.g., health care practitioners). Accordingly, subjects at risk ofbecoming infected by a pathogen that has a receptor are identified andthen are provided a prophylactically effective amount of specificityexchanger.

One prophylactic application for the specificity exchangers describedherein concerns coating or cross-linking the specificity exchanger to amedical device or implant. Implantable medical devices tend to serve asfoci for infection by a number of bacterial species. Suchdevice-associated infections are promoted by the tendency of theseorganisms to adhere to and colonize the surface of the device.Consequently, there is a considerable need to develop surfaces that areless prone to promote the adverse biological reactions that typicallyaccompany the implantation of a medical device.

By one approach, the medical device is coated in a solution ofcontaining a specificity exchanger. Prior to implantation, medicaldevices (e.g., a prosthetic valve) can be stored in a solution ofspecificity exchangers, for example. Medical devices can also be coatedin a powder or gel having a specificity exchanger. For example, gloves,condoms, and intrauterine devices can be coated in a powder or gel thatcontains a specificity exchanger that interacts with a bacterial orviral receptor. Once implanted in the body, these specificity exchangersprovide a prophylactic barrier to infection by a pathogen.

In some embodiments, the specificity exchanger is immobilized to themedical device. As described above, the medical device is a support towhich a specificity exchanger can be attached. Immobilization may occurby hydrophobic interaction between the specificity exchanger and themedical device but a preferable way to immobilize a specificityexchanger to a medical device involves covalent attachment. For example,medical devices can be manufactured with a reactive group that interactswith a reactive group present on the specificity exchanger.

By one approach, a periodate is combined with a specificity exchangercomprising a 2-aminoalcohol moiety to form an aldehyde-functionalexchanger in an aqueous solution having a pH between about 4 and about 9and a temperature between about 0 and about 50 degrees Celsius. Next,the aldehyde-functional exchanger is combined with the biomaterialsurface of a medical device that comprises a primary amine moiety toimmobilize the specificity exchanger on the support surface through animine moiety. Then, the imine moiety is reacted with a reducing agent toform an immobilized specificity exchanger on the biomaterial surfacethrough a secondary amine linkage. A similar chemistry can be used toattach the sugar to the support and/or specificity domain of thespecificity exchanger, as well. Other approaches for cross-linkingmolecules to medical devices, (such as described in U.S. Pat. No.6,017,741, herein expressly incorporated by reference in its entirety),can be modified to immobilize the specificity exchangers describedherein.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims. All referencescited herein are hereby expressly incorporated by reference in theirentireties.

1. An isolated glycoconjugate peptide comprising an HIV gp120 bindingfragment of CD4 synthetically conjugated to gal α (1,3) gal β, whereinsaid fragment is less than 20 amino acids in length.
 2. Theglycoconjugate peptide of claim 1, wherein the sequence of said fragmentis selected from the group consisting of SEQ. ID. NO. 2, SEQ. ID. NO. 3,SEQ. ID. NO. 4, SEQ. ID. NO.
 5. SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID.NO. 8, and SEQ. ID. NO.
 9. 3. The isolated glycoconjugate peptide ofclaim 1, wherein said glycoconjugate peptide is linear.
 4. The isolatedglycoconjugate peptide of claim 1, wherein said gal α (1,3) gal β issynthetically conjugated to said HIV gp120 binding fragment of CD4 byattachment at one amino acid.
 5. The isolated glycoconjugate peptide ofclaim 4, wherein said gal α (1,3) gal β is synthetically conjugated to ahydroxylated amino acid.
 6. The isolated glycoconjugate peptide of claim4, wherein said gal α (1,3) gal β is synthetically conjugated by anNH₂-linkage.
 7. The isolated glycoconjugate peptide of claim 4, whereinsaid gal α (1,3) gal β is synthetically conjugated to the N-terminal endof the HIV gp120 binding fragment of CD4.
 8. The isolated glycoconjugatepeptide of claim 1, wherein said gal α (1,3) gal β is syntheticallyconjugated to a hydioxylated amino acid.
 9. The isolated glycoconjugatepeptide of claim 1, wherein said gal α (1,3) gal β is syntheticallyconjugated by an NH₂-linkage.
 10. The isolated glycoconjugate peptide ofclaim 1, wherein said gal α (1,3) gal β is synthetically conjugated tothe N-terminal end of the HIV gp120 binding fragment of CD4.